Background: Pre-exposure prophylaxis (PrEP) is only effective in preventing new HIV infections when taken consistently. In clinical practice, asking a patient about their adherence (self-report) is the predominant method of assessing adherence to PrEP. Although inexpensive and noninvasive, self-report is subject to social desirability and recall biases. Several clinical trials demonstrate a discrepancy between self-reported adherence and biomarker-based recent adherence. Less is known about the accuracy of self-report in real-world clinical settings. This brief report addresses this knowledge gap and describes the concordance between self-reported adherence and biomarker-based adherence in real-world clinical settings. Methods: A liquid chromatography–mass spectrometry urine test for tenofovir was developed and used clinically to detect recent nonadherence (no dose in at least 48 hours) for each individual. Two clinics' standard operating procedures recommend utilization of the urine-based adherence test for patients who self-report that they are not struggling with adherence. Those who self-report struggling with adherence receive enhanced adherence support without the need for additional testing. The number of results indicating recent nonadherence from these 2 clinics were analyzed to assess the concordance between self-reported adherence and biomarker-based adherence. Results: Across 2 clinics, 3987 tests were conducted from patients self-reporting as “adherent,” and 564 [14.1%; 95% confidence interval (CI): 13.1% to 15.2%] demonstrated recent nonadherence with the liquid chromatography–mass spectrometry test. At clinic #1 in Florida, 3200 tests were conducted, and 465 (14.5%; 95% CI: 13.3% to 15.8%) demonstrated recent nonadherence. At clinic #2 in Texas, 787 tests were conducted, and 99 (12.6%; 95% CI: 10.4% to 14.9%) demonstrated recent nonadherence. Conclusions: Utilization of biomarker-based adherence monitoring at these 2 clinics resulted in 564 additional patients receiving enhanced adherence support who otherwise would not have been identified as nonadherent to their prescribed PrEP regimen. These findings suggest that objective adherence monitoring can be used clinically to enable providers to identify nonadherent patients and allocate support services accordingly.