Objective: In this study, we aimed to evaluate the validity and reliability of the free oral pre-exposure prophylaxis (PrEP) adherence questionnaire among patients with sexually transmitted infection who do not have HIV infection in Shanghai. Methods: We conducted a cross-sectional study among 816 patients from sexually transmitted infection (STI) clinics in Shanghai. The questionnaire included self-reported demographic characteristics, items on adherence to free oral pre-exposure prophylaxis (PrEP), and PrEP uptake behavior measurement. Item analysis was carried out by calculating the critical ratio and item-total correlations. Internal consistency and split-half reliability were measured to assess reliability. Content validity, construct validity, convergent validity and discriminant validity were measured to assess validity. We conducted logistic regression with receiver operating characteristic (ROC) curve analysis to assess the predictive value of the adherence questionnaire for PrEP uptake behavior. Results: All items were considered acceptable in the item analysis (P<0.05). The questionnaire had a Cronbach’s alpha of 0.757 and split-half reliability of 0.633. 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 with all factors loading more than 0.4, which was validated in confirmatory factor analysis (c2=47.1, degrees of freedom=14, root mean square error of approximation=0.078). According to regression analysis, adding PrEP adherence questionnaire scores contributed to prediction of PrEP uptake behavior (P<0.001). The maximum area under the ROC curve was 0.778 (95% confidence interval: 0.739–0.817), corresponding to a cutoff score of 9.Conclusion: The PrEP adherence questionnaire generally has acceptable reliability and validity. This questionnaire can be used as a tool to evaluate PrEP adherence in patients with STI who do not have HIV infection.