Adolescents in the United States (US) continue to be disproportionately impacted by HIV and STIs. We investigated the associations between sexual health and substance use behaviors with HIV and STI testing among high school students in the US. Cross-sectional weighted stepwise multivariate logistic regression models were conducted to determine the odds of lifetime HIV and STI testing among students, stratified by sex. Drugs and alcohol before sex, condom use during sex, number of sex partners, sex of sexual partners, substance use (e-cigarette, alcohol, and cannabis) frequency, lifetime cocaine use, and lifetime prescription drug misuse were predictors, adjusting for race/ethnicity and age. This analysis used the 2019 and 2021 cycles of the CDC Youth Risk Behavioral Surveillance System (YRBSS) data. Our sample of adolescents (n = 30,909) had a mean age of 16
51% were male. Females engaging in substance use had increased odds of HIV and STI testing whereas that pattern did not reflect among males. Multiracial male adolescents had increased odds of HIV testing. Females engaging in condomless sex had lower odds of HIV and STI testing. This study highlights the need for tailored HIV and STI testing promotion and ongoing efforts for dismantling barriers to testing services. Furthermore, our study suggests that screenings follow the concurrent nature of sexual health and substance use behaviors of adolescents for integrated HIV/STI testing.