Background: Studies of condom efficacy rely on self-reported behavior. Objective markers of exposure to semen may provide a more valid assessment of condom failure and failure to use condoms. Goals of this Study: To compare three semen biomarkers: acid phosphatase (AP) activity, prostate specific antigen (PSA), and the human seminal plasma antigen (MHS-5). Study Design: Twenty women were intravaginally inoculated with six measured, increasingly larger amounts of their partners' semen. Vaginal fluid was collected by the participant using swabs and tested. Results: Background levels of PSA were low (0.00-1.25 ng/ml), background levels of AP were variable (0-350 U/I), and all preinoculation samples were negative for MHS-5. All postinoculation samples were positive for PSA, 64 of 117 (55%) for AP, and 14 of 120 (12%) for MHS-5. Conclusion: The PSA immunoassay was the best semen biomarker under these sampling and testing conditions.