Introduction: Young men who have sex with men (YMSM) and young transgender women (YTGW) in Thailand are at high HIV risk. HIV self-tests (HIVSTs) are rapidly administrable and prompt linkage to HIV treatment or prevention services. This study assesses the acceptability and feasibility of blood-based HIVST use in adolescents. Methods: A cross-sectional study was conducted among YMSM and YTGW aged 15–19 years with HIV acquisition risk. Participants completed questionnaires on the HIVST and then administered INSTI® independently, an HIVST immunoassay detecting gp41 and gp36 antibodies from finger-stick blood. Confirmatory HIV antibody tests were performed. Results: Between July and September 2020, 90 adolescents were enrolled. Mean (SD) age was 17.6 (1.1) years. Half ( N = 45) were YMSM. Forty-six (51%) were first-time HIV testers, and 32 (36%) had “ever used” HIV pre-exposure prophylaxis (PrEP). Two (2.2%, 95% CI: 0.0–5.3) tested positive, 21 (23.4%) invalid, and 67 (74.4%) negative. Invalidity causes included 17 (81%) insufficient blood, 3 (14%) buffer spillage, and 1 (5%) procedural missteps; all had negative HIV antibody tests. HIV self-test acceptability was 87.8% (95% CI: 81.0–94.5). Most (79%) preferred HIVST performance in hospital rather than at home. Conclusions: HIVSTs are acceptable in HIV at-risk adolescents. Blood-based HIVSTs should be positioned as rapid point-of-care tests with real-time linkage to HIV services.