BACKGROUND: In Alabama, the undiagnosed HIV rate is over 20%; youth and young adults, particularly those who identify as sexual and gender minority individuals, are at elevated risk for HIV acquisition and are the only demographic group in the United States with rising rates of new infections. Adolescence is a period marked by exploration, risk taking, and learning, making comprehensive sexual health education a high-priority prevention strategy for HIV and sexually transmitted infections. However, in Alabama, school-based sexual health and HIV prevention education is strictly regulated and does not address the unique needs of sexual and gender minority teenagers. OBJECTIVE: To understand knowledge gaps related to sexual health, HIV prevention, and pre-exposure prophylaxis (PrEP), we conducted the Alabama Youth Survey with individuals aged 14-17 years. In the survey, we also evaluated young sexual and gender minority individuals' preferences related to prevention modalities and trusted sources of health information. METHODS: Between September 2023 and March 2024, we conducted a web-based survey with 14- to 17-year-olds who are assigned male at birth, are sexually attracted to male youth, and lived in Alabama. Half of the study's participants were recruited through community partners, the Magic City Acceptance Academy and Magic City Acceptance Center. The other half were recruited on the web via social media. A 7-step fraud and bot detection protocol was implemented and applied to web-based recruitment to reduce the likelihood of collecting false information. Once data are ready, we will compute frequencies for each measure and construct summary scores of scales, such as HIV and PrEP knowledge, to determine internal consistency. Using multivariable logistic regression, we will examine associations between personal characteristics of survey respondents and key constructs using SPSS 29 (IBM Corp) or SAS 9.4 (SAS Institute). RESULTS: Analyses are ongoing (N=206) and will conclude in June 2025. Preliminary results include a sample mean age of 16.21 (SD 0.88) years; about a quarter identified as transgender or gender nonconforming, with 6% stating their gender as a transgender woman. A total of 30% self-reported their race as African American or Black; 12% were Hispanic or Latinx. More than half reported being sexually active in the past 6 months. Primary data analyses will be completed in mid-2025. If findings are promising, results will be used as preliminary data to support the development of an intervention to address knowledge gaps and prevention preferences. CONCLUSIONS: If the study is successful, it will yield information on HIV knowledge, PrEP awareness, PrEP preferences, and related outcomes among sexual and gender minority teenagers in Alabama, an underserved, hard-to-reach, but also high-priority population for public health efforts to Ending the HIV Epidemic. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/63114.