BACKGROUND: Mobile health (mHealth) apps have the potential to be a useful mode of delivering HIV prevention information, particularly for young men (13-24 years) who account for 21% of new HIV diagnoses in the United States. We translated an existing evidence-based, face-to-face HIV prevention curriculum into a portable platform and developed a mobile Web app: MyPEEPS Mobile. OBJECTIVE: The purpose of this study was to assess the usability of MyPEEPS Mobile from both expert and end user perspectives. METHODS: We conducted a heuristic evaluation with five experts in informatics to identify violations of usability principles and end user usability testing with 20 young men aged 15 to 18 years in New York, NY, Birmingham, AL, and Chicago, IL to identify potential obstacles to their use of the app. RESULTS: Mean scores of the overall severity of the identified heuristic violations rated by experts ranged from 0.4 and 2.6 (0=no usability problem to 4=usability catastrophe). Overall, our end users successfully completed the tasks associated with use case scenarios and provided comments/recommendations on improving usability of MyPEEPS Mobile. The mean of the overall Post-Study System Usability Questionnaire scores rated by the end users was 1.63 (SD 0.65), reflecting strong user acceptance of the app. CONCLUSIONS: The comments made by experts and end users will be used to refine MyPEEPS Mobile prior to a pilot study assessing the acceptability of the app across diverse sexual minority young men in their everyday lives.