Background: Youth who are experiencing homelessness face a higher risk of HIV infection compared to their housed peers, and suicide and overdose remain the leading causes of death among homeless youth. Just-in-Time Adaptive Interventions (JITAIs) are gaining momentum for HIV prevention and substance use research. Yet, most interventions for homeless youth have not addressed modifiable real-time factors. Objective: This paper describes the development and implementation of a randomized attention-controlled trial to assess the efficacy of motivating youth to reduce infections, disconnections, and emotional dysregulation (MY-RIDE), a JITAI to improve HIV prevention and substance use in homeless youth. Methods: This study will enroll 320 homeless youth aged 18-25 years. The intervention was co-designed with homeless youth using the Information-Motivation-Behavioral Skills Model and consists of an individual nurse-led session about HIV prevention and 3 months of a JITAI with personalized messaging delivered by phone in real time in response to one’s current level of risk. Participants also had access to an on-demand nurse helpline through the app. Results: Institutional review board approval was obtained in the summer of 2024. Recruitment began in the fall of 2024 at shelters, drop-in centers, and other organizations that serve homeless youth. Participants complete a baseline survey and HIV/sexually transmitted infection (STI) testing and are provided with a smartphone with the intervention app. Follow-up surveys and HIV/STI testing are conducted at immediate, 3-, 6-, and 12-month time points post intervention to assess uptake of HIV prevention strategies and substance use reduction. A total of 192 are enrolled to date. Conclusions: The results of this study will determine whether MY-RIDE increases HIV prevention strategies and decreases substance use when compared to homeless youth in the attention control group. We will also evaluate if MY-RIDE impacts protective factors such as willingness to take pre-exposure prophylaxis medication and use of mental health and substance use services, and antecedents of risk such as stress, substance use urge, and substance use. Trial Registration: Clinicaltrials.gov NCT06074354; https://clinicaltrials.gov/study/NCT06074354