Specific age groups, persons who participate in risky sexual behavior, or those have certain health conditions may require screening. The CDC recommends that sexually active women under the age of 25 and those over 25 at risk should be screened for chlamydia and gonorrhea yearly. Appropriate times for screening are during regular pelvic examinations and preconception evaluations. Nucleic acid amplification tests are the recommended method of diagnosis for gonorrhea and chlamydia. This can be done on either urine in both men and women, vaginal or cervical swabs in women, or urethral swabs in men. Screening can be performed:

- to assess the presence of infection and prevent tubal infertility in women

- during the initial evaluation before infertility treatment

- to identify HIV infection

- for men who have sex with men

- for those who may have been exposed to hepatitis C

- for HCV

Testing may be for a single infection, or consist of a number of tests for a range of STIs, including tests for syphilis, trichomonas, gonorrhea, chlamydia, herpes, hepatitis and HIV. No procedure tests for all infectious agents.

STI tests may be used for a number of reasons:

- as a diagnostic test to determine the cause of symptoms or illness

- as a screening test to detect asymptomatic or presymptomatic infections

- as a check that prospective sexual partners are free of disease before they engage in sex without safer sex precautions (for example, when starting a long term mutually monogamous sexual relationship, in fluid bonding, or for procreation).

- as a check prior to or during pregnancy, to prevent harm to the baby

- as a check after birth, to check that the baby has not caught an STI from the mother

- to prevent the use of infected donated blood or organs

- as part of the process of contact tracing from a known infected individual

- as part of mass epidemiological surveillance

Early identification and treatment results in less chance to spread disease, and for some conditions may improve the outcomes of treatment. There is often a window period after initial infection during which an STI test will be negative. During this period, the infection may be transmissible. The duration of this period varies depending on the infection and the test. Diagnosis may also be delayed by reluctance of the infected person to seek a medical professional. One report indicated that people turn to the Internet rather than to a medical professional for information on STIs to a higher degree than for other sexual problems.

For sexually active women who are not pregnant, screening is recommended in those under 25 and others at risk of infection. Risk factors include a history of chlamydial or other sexually transmitted infection, new or multiple sexual partners, and inconsistent condom use. For pregnant women, guidelines vary: screening women with age or other risk factors is recommended by the U.S. Preventive Services Task Force (USPSTF) (which recommends screening women under 25) and the American Academy of Family Physicians (which recommends screening women aged 25 or younger). The American College of Obstetricians and Gynecologists recommends screening all at risk, while the Centers for Disease Control and Prevention recommend universal screening of pregnant women. The USPSTF acknowledges that in some communities there may be other risk factors for infection, such as ethnicity. Evidence-based recommendations for screening initiation, intervals and termination are currently not possible. For men, the USPSTF concludes evidence is currently insufficient to determine if regular screening of men for chlamydia is beneficial. They recommend regular screening of men who are at increased risk for HIV or syphilis infection.

In the United Kingdom the National Health Service (NHS) aims to:

1. Prevent and control chlamydia infection through early detection and treatment of asymptomatic infection;

2. Reduce onward transmission to sexual partners;

3. Prevent the consequences of untreated infection;

4. Test at least 25 percent of the sexually active under 25 population annually.

5. Retest after treatment.

The United States Preventive Services Task Force (USPSTF) recommends screening for gonorrhea in women at increased risk of infection, which includes all sexually active women younger than 25 years. Extragenital gonorrhea and chlamydia are highest in men who have sex with men (MSM). Additionally, the USPSTF also recommends routine screening in people who have previously tested positive for gonorrhea or have multiple sexual partners and individuals who use condoms inconsistently, provide sexual favors for money, or have sex while under the influence of alcohol or drugs.

Screening for gonorrhea in women who are (or intend to become) pregnant, and who are found to be at high risk for sexually transmitted diseases, is recommended as part of prenatal care in the United States.

Traditionally, gonorrhea was diagnosed with gram stain and culture; however, newer polymerase chain reaction (PCR)-based testing methods are becoming more common. In those failing initial treatment, culture should be done to determine sensitivity to antibiotics. All people testing positive for gonorrhea should be tested for other sexually transmitted diseases such as chlamydia, syphilis, and human immunodeficiency virus.

The diagnosis of genital chlamydial infections evolved rapidly from the 1990s through 2006. Nucleic acid amplification tests (NAAT), such as polymerase chain reaction (PCR), transcription mediated amplification (TMA), and the DNA strand displacement amplification (SDA) now are the mainstays. NAAT for chlamydia may be performed on swab specimens sampled from the cervix (women) or urethra (men), on self-collected vaginal swabs, or on voided urine. NAAT has been estimated to have a sensitivity of approximately 90% and a specificity of approximately 99%, regardless of sampling from a cervical swab or by urine specimen. In women seeking an STI clinic and a urine test is negative, a subsequent cervical swab has been estimated to be positive in approximately 2% of the time.

At present, the NAATs have regulatory approval only for testing urogenital specimens, although rapidly evolving research indicates that they may give reliable results on rectal specimens.

Because of improved test accuracy, ease of specimen management, convenience in specimen management, and ease of screening sexually active men and women, the NAATs have largely replaced culture, the historic gold standard for chlamydia diagnosis, and the non-amplified probe tests. The latter test is relatively insensitive, successfully detecting only 60–80% of infections in asymptomatic women, and often giving falsely positive results. Culture remains useful in selected circumstances and is currently the only assay approved for testing non-genital specimens. Other method also exist including: ligase chain reaction (LCR), direct fluorescent antibody resting, enzyme immunoassay, and cell culture.

Use of male condoms or female condoms may help prevent the spread of trichomoniasis, although careful studies have never been done that focus on how to prevent this infection. Infection with Trichomoniasis through water is unlikely because "Trichomonas vaginalis" dies in water after 45–60 minutes, in thermal water after 30 minutes to 3 hours and in d urine after 5–6 hours.

Currently there are no routine standard screening requirements for the general U.S. population receiving family planning or STI testing. The Centers for Disease Control and Prevention (CDC) recommends Trichomoniasis testing for females with vaginal discharge and can be considered for females at higher risk for infection or of HIV-positive serostatus.

The advent of new, highly specific and sensitive trichomoniasis tests present opportunities for new screening protocols for both men and women. Careful planning, discussion, and research are required to determine the cost-efficiency and most beneficial use of these new tests for the diagnosis and treatment of trichomoniasis in the U.S., which can lead to better prevention efforts.

A number of strategies have been found to improve follow-up for STI testing including email and text messaging as reminders of appointments.

There are three main ways to test for Trichomoniasis.

- The first is known as saline microscopy. This is the most commonly used method and requires an endocervical, vaginal, or penile swab specimen for examination under a microscope. The presence of one or multiple trichomonads constitutes a positive result. This method is cheap but has a low sensitivity (60-70%) often due to an inadequate sample, resulting in false negatives.

- The second diagnostic method is culture, which has historically been the "gold standard" in infectious disease diagnosis. Trichomonas Vaginalis culture tests are relatively cheap; however, sensitivity is still somewhat low (70-89%).

- The third method includes the nucleic acid amplification tests (NAATs) which are more sensitive. These tests are more costly than microscopy and culture, and are highly sensitive (80-90%).

Syphilis is difficult to diagnose clinically early in its presentation. Confirmation is either via blood tests or direct visual inspection using microscopy. Blood tests are more commonly used, as they are easier to perform. Diagnostic tests are unable to distinguish between the stages of the disease.

The CDC recommends that sexually active men who have sex with men be tested at least yearly. The USPSTF also recommends screening among those at high risk.

Syphilis is a notifiable disease in many countries, including Canada the European Union, and the United States. This means health care providers are required to notify public health authorities, which will then ideally provide partner notification to the person's partners. Physicians may also encourage patients to send their partners to seek care. Several strategies have been found to improve follow-up for STI testing, including email and text messaging as reminders of appointments.

There are several methods to diagnose meningeal syphilis. One of the most common ways include visualizing the organisms by immunofluorescence and dark field microscopy. Dark field microscopy initially had the finding that the spirochete has a corkscrew appearance and that it is spirillar and gram (-) bacteria. Another method would also be through the screening test and serology. Serology includes two types of antibody test: Nontreponemal antibody test and Treponemal antibody test (specific test). The Nontreponemal antibody test screens with VDRL (Venereal Disease Research Lab) and RPR (Rapid Plasma Reagin). The Treponemal antibody test (specific test) confirms with FTA-ABS (Fluorescent treponemal antibody-absorption). Brain imaging and MRI scans may be used when diagnosing patients; however, they do not prove to be as effective as specific tests. Specific tests for treponemal antibody are typically more expensive because the earliest anitbodies bind to spirochetes. These tests are usually more specific and remain positive in patients with other treponemal diseases.

The most popular treatment forms for any type of syphilis uses penicillin, which has been an effective treatment used since the 1940s.

Other forms also include Benzathine penicillin, which is usually used for primary and secondary syphilis (it has no resistance to penicillin however). Benzathine penicillin is used for long acting form, and if conditions worsen, penicillin G is used for late syphilis.

From bubo pus or ulcer secretions, "H. ducreyi" can be identified. PCR-based identification of organisms is available. Simple, rapid, sensitive and inexpensive antigen detection methods for "H. ducreyi" identification are also popular. Serologic detection of "H. ducreyi" is and uses outer membrane protein and lipooligosaccharide.

Despite many distinguishing features, the clinical spectrums of following diseases may overlap with chancroid:

- Primary syphilis

- Genital herpes

Practical clinical approach for this STI as Genital Ulcer Disease is to rule out top differential diagnosis of Syphilis and Herpes and consider empirical treatment for Chancroid as testing is not commonly done for the latter.

When physical examination of the newborn shows signs of a vertically transmitted infection, the examiner may test blood, urine, and spinal fluid for evidence of the infections listed above. Diagnosis can be confirmed by culture of one of the specific pathogens or by increased levels of IgM against the pathogen.

Although it is possible to test for HPV DNA in other kinds of infections, there are no FDA-approved tests for general screening in the United States or tests approved by the Canadian government, since the testing is inconclusive and considered medically unnecessary.

Genital warts are the only visible sign of low-risk genital HPV and can be identified with a visual check. These visible growths, however, are the result of non-carcinogenic HPV types. Five percent acetic acid (vinegar) is used to identify both warts and squamous intraepithelial neoplasia (SIL) lesions with limited success by causing abnormal tissue to appear white, but most doctors have found this technique helpful only in moist areas, such as the female genital tract. At this time, HPV test for males are used only in research.

Research into testing for HPV by antibody presence has been done. The approach is looking for an immune response in blood, which would contain antibodies for HPV if the patient is HPV positive. The reliability of such tests hasn't been proven, as there hasn't been a FDA approved product as of March 2014; testing by blood would be a less invasive test for screening purposes.

Research studies have tested for and found HPV, including high-risk types (i.e. the types found in cancers), on fingers, mouth, saliva, anus, urethra, urine, semen, blood, scrotum and penis. However, most research tests have used Dacron swabs and custom analysis not available to the general public.

A Brazilian study used the readily available Qiagen/Digene test mentioned above (off label) to test men's penis, scrotum and anus. Each of the 50 men had been a partner for at least 6 months of a woman who was positive for high-risk HPV. They found high-risk HPV on 60% of these men, primarily the penis. "The specimens were obtained using a vigorous motion of the conical brush included in the Digene kit after spraying the anogenital region with saline solution."

A slightly different method also used cytobrushes (but custom lab analysis) and found 37% of 582 Mexican army recruits positive for high risk HPV. They were told not to wash genitals for 12 hours before sampling. (Other studies are silent on washing, a particular gap in studies of hands). They included the urethra as well as scrotum and penis, but the urethra added less than 1% to the HPV rate. Studies like this led Giuliano to recommend sampling the glans, shaft and crease between them and scrotum, since sampling the urethra or anus added very little to diagnosis. Dunne recommends glans, shaft, their crease, and foreskin.

A small study of cytobrushes on 10 US men where the brush was wet, rather than the skin, found 2 of 10 men were positive for HPV (type not reported). Their lab analysis was not the same as either study above. This small study found 4 of 10 men positive for HPV when the skin was rubbed with 600 grit emery paper, then swabbed with a wet Dacron swab. Since emery paper and brush were analyzed together at the lab, it is not known if the emery paper collected viruses or loosened them for the swab to collect.

Studies have found collection by men from their own skin (with emery paper and Dacron swabs) as effective as by a clinician, sometimes more so, since patients were more willing to scrape vigorously.

Other studies have used similar cytobrushes to sample fingertips and under the fingernails, though without wetting the area or brush.

Other studies analyzed urine, semen, and blood and found varying amounts of HPV, but there is no publicly available test for them.

There is not a wide range of tests even though HPV is common. Clinicians depend on the vaccine among young people and high clearance rates (see Clearance subsection in Virology) to create a low risk of disease and mortality, and treat the cancers when they appear. Others believe that reducing HPV infection in more men and women, even when it has no symptoms, is important (herd immunity) to prevent more cancers rather than just treating them. Where tests are used, negative test results show safety from transmission, and positive test results show where shielding (condoms, gloves) is needed to prevent transmission until the infection clears.

HIV/AIDS is diagnosed via laboratory testing and then staged based on the presence of certain signs or symptoms. HIV screening is recommended by the United States Preventive Services Task Force for all people 15 years to 65 years of age including all pregnant women. Additionally, testing is recommended for those at high risk, which includes anyone diagnosed with a sexually transmitted illness. In many areas of the world, a third of HIV carriers only discover they are infected at an advanced stage of the disease when AIDS or severe immunodeficiency has become apparent.

Some vertically transmitted infections, such as toxoplasmosis and syphilis, can be effectively treated with antibiotics if the mother is diagnosed early in her pregnancy. Many viral vertically transmitted infections have no effective treatment, but some, notably rubella and varicella-zoster, can be prevented by vaccinating the mother prior to pregnancy.

If the mother has active herpes simplex (as may be suggested by a pap test), delivery by Caesarean section can prevent the newborn from contact, and consequent infection, with this virus.

IgG antibody may play crucial role in prevention of intrauterine infections and extensive research is going on for developing IgG-based therapies for treatment and vaccination.

Most people infected with HIV develop specific antibodies (i.e. seroconvert) within three to twelve weeks of the initial infection. Diagnosis of primary HIV before seroconversion is done by measuring HIV-RNA or p24 antigen. Positive results obtained by antibody or PCR testing are confirmed either by a different antibody or by PCR.

Antibody tests in children younger than 18 months are typically inaccurate due to the continued presence of maternal antibodies. Thus HIV infection can only be diagnosed by PCR testing for HIV RNA or DNA, or via testing for the p24 antigen. Much of the world lacks access to reliable PCR testing and many places simply wait until either symptoms develop or the child is old enough for accurate antibody testing. In sub-Saharan Africa as of 2007–2009 between 30 and 70% of the population were aware of their HIV status. In 2009, between 3.6 and 42% of men and women in Sub-Saharan countries were tested which represented a significant increase compared to previous years.

The diagnosis of genital warts is most often made visually, but may require confirmation by biopsy in some cases. Smaller warts may occasionally be confused with molluscum contagiosum.

Genital warts, histopathologically, characteristically rise above the skin surface due to enlargement of the dermal papillae, have parakeratosis and the characteristic nuclear changes typical of HPV infections (nuclear enlargement with perinuclear clearing).

DNA tests are available for diagnosis of high-risk HPV infections. Because genital warts are caused by low-risk HPV types, DNA tests cannot be used for diagnosis of genital warts or other low-risk HPV infections.

Some practitioners use an acetic acid solution to identify smaller warts ("subclinical lesions"), but this practice is controversial. Because a diagnosis made with acetic acid will not meaningfully affect the course of the disease, and cannot be verified by a more specific test, a 2007 UK guideline advises against its use.

Testing peoples blood, including those who are pregnant, who do not have symptoms for HSV is not recommended. This is due to concerns of greater harm than benefit such as relationship problems in the setting of a high rate of tests that may be falsely positive.

The diagnosis usually is made serologically (through complement fixation) and by exclusion of other causes of inguinal lymphadenopathy or genital ulcers. Serologic testing has a sensitivity of 80% after 2 weeks. Serologic testing may not be specific for serotype (has some cross reactivity with other chlamydia species) and can suggest LGV from other forms because of their difference in dilution, 1:64 more likely to be LGV and lower than 1:16 is likely to be other chlamydia forms (emedicine).

For identification of serotypes, culture is often used. Culture is difficult. Requiring a special medium, cycloheximide-treated McCoy or HeLa cells, and yields are still only 30-50%. DFA, or direct fluorescent antibody test, PCR of likely infected areas and pus, are also sometimes used. DFA test for the L-type serovar of C trachomatis is the most sensitive and specific test, but is not readily available.

If polymerase chain reaction (PCR) tests on infected material are positive, subsequent restriction endonuclease pattern analysis of the amplified outer membrane protein A gene can be done to determine the genotype.

Recently a fast realtime PCR (TaqMan analysis) has been developed to diagnose LGV. With this method an accurate diagnosis is feasible within a day. It has been noted that one type of testing may not be thorough enough.

Diagnosis is made by taking samples for bacterial culture from all accessible sites. In mares, this includes the endometrium, cervix, clitoral fossa and sinuses. In stallions, samples are taken from the skin folds of the prepuce, urethral fossa, urethra, and the pre-ejaculatory fluid. Samples are refrigerated and transported to an approved testing laboratory within 48 hours of collection.

Blood tests for mares are available for detecting antibodies to "Taylorella equigenitalis". Blood tests are not possible for stallions. These tests become positive 10 or more days after infection. If positive, they only indicate that the mare has had the disease in the past, and do not indicate whether the mare is a carrier now.

Genital herpes can be spread by viral shedding prior to and following the visual signs of symptoms.