HSV testing is used to detect the presence of the herpes simplex virus in those who have genital sores, encephalitis, and in newborns suspected of having neonatal herpes (a rare but serious condition where herpes is contracted during birth). A pregnant woman who has been diagnosed with herpes may be monitored regularly prior to delivery to identify a reactivation of her infection (which would indicate the necessity for a caesarean section to avoid infecting the baby). The primary methods of testing for the virus are the herpes culture and HSV DNA testing.

Although it is not as sensitive, HSV antibody testing can be used to help diagnose an acute HSV infection if acute and convalescent blood samples are collected. The convalescent blood sample is collected several weeks after the acute sample, and HSV IgG antibody levels are compared to see if they have risen significantly (indicating a current infection). Antibody testing may also be used to screen certain populations, such as sexually active people, potential organ transplant recipients, and those with HIV/AIDS, for a previously contracted HSV infection.

A herpes culture (or HSV DNA testing) may be ordered when a patient has a blister or vesicle on their genitals. HSV DNA testing is ordered when a patient has encephalitis that the doctor suspects may be caused by a virus. HSV testing may be ordered regularly when a pregnant woman has herpes. A mother and newborn may be tested for HSV when a baby shows signs of HSV infection (such as meningitis or skin lesions).

HSV antibody testing is ordered primarily when a patient is being screened for a previous exposure to HSV. Occasionally, acute and convalescent HSV antibody testing may be ordered when a current infection is suspected.

A positive herpes simplex culture or HSV DNA test from a vesicle scraping indicates an active HSV-1 or HSV-2 infection. A negative test result indicates that the herpes simplex virus was not isolated but does not definitely rule out the presence of virus. This is because if the specimen taken does not contain actively replicating virus or if the sample was not transported under optimum conditions, no viable virus may be detectable, resulting in a false negative result. For example, viruses can be readily inactivated and if the sample was taken from an older lesion, not a fresh blister, or if transport of the sample was delayed, there may not be sufficient virus to detect even though the patient is infected.

The presence of HSV-1 or HSV-2 IgM antibodies indicates an active or recent infection. HSV-1 or HSV-2 IgG antibodies indicate a previous infection. A significant increase in HSV IgG antibodies, measured by comparing acute and convalescent samples, indicates an active or recent infection. Negative HSV antibody results mean that it is unlikely that the patient has been exposed to HSV, or that the body has not had time to begin HSV antibody production.

The most serious, or life-threatening, HSV infections can occur in newborns after perinatal infection and in immunocompromised individuals. The lesions tend to be more extensive and persist longer than in immunocompetent individuals. Infection with HSV can increase HIV viral load. HSV-2 infection is a significant opportunistic infection in HIV-infected individuals; up to 90% of HIV-infected individuals are co-infected with HSV-2.

HSV, in combination with human papilloma virus (HPV) infection, has been associated with a higher risk of developing cervical cancer.