In the United States, young men who have sex with men (YMSM) and young transgender women (YTGW) are disproportionality affected by HIV. To overcome this public health problem, we created and tested the mLab application (app), a novel mobile health (mHealth) that offers HIV prevention information and an imaging algorithm for interpreting the at-home HIV test. This study assessed the mLab app usability for HIV testing and its relation to users’ education and health literacy. The results showed high user satisfaction and perceived usability of mLab to provide accessible HIV testing solutions for YMSM and YTGW. Findings suggest that the rigorous user-centered design of the mLab app supported a usable app independent of education and health literacy levels.