BACKGROUND: Ensuring adherence guarantees the efficacy of pre-exposure prophylaxis (PrEP). METHODS: We conducted a cross-sectional study among 816 sexually transmitted infection (STI) patients in Shanghai. The questionnaire included self-reported demographic characteristics, self-administered items on adherence to free oral PrEP, and PrEP uptake behavior measurement. We conducted item analysis, reliability analysis, validity analysis and receiver operating characteristic (ROC) curve analysis. RESULTS: Not all items were considered acceptable in the item analysis. The questionnaire had a McDonald's ω coefficient of 0.847. The scale-level content validity index (CVI) was 0.938 and the item-level CVI of each item ranged from 0.750 to 1. In exploratory factor analysis, we introduced a four-factor model accounting for 79.838% of the aggregate variance, which was validated in confirmatory factor analysis. Adding PrEP adherence questionnaire scores contributed to prediction of PrEP uptake behavior ( < 0.001) in regression analysis. The maximum area under the ROC curve was 0.778 (95% IC: 0.739-0.817). CONCLUSION: The PrEP adherence questionnaire presented psychometric validation among STI patients.