Biomarkers of vaginal exposure to semen, long used in forensic medicine, are now becoming important in the development of vaginal microbicides to prevent HIV/STIs and the development of contraceptives. Semen biomarkers could help evaluate the safety of a new physical or chemical barrier, give preliminary indication of the effectiveness of physical barriers such as diaphragms or condoms, and provide information on unprotected intercourse among participants in a clinical trial who have been advised to use condoms. Candidate biomarkers of semen exposure fall into two broad categories: (1) biomarkers of seminal plasma, among which prostate-specific antigen (PSA) is the best characterized; and (2) biomarkers of spermatozoa and other cells present in semen. This paper, authored by a working group of investigators performing research in the field of semen biomarkers, summarizes the characteristics of an ideal semen biomarker, reviews preclinical and clinical data on existing and potential biomarkers, and outlines the steps that should be carried out to develop an improved biomarker of semen exposure.