Background The Theoretical Framework of Acceptability (TFA) provides a comprehensive lens for assessing acceptability of healthcare interventions. While the TFA has seven constructs, limited literature exists on its psychometric properties in low-resource settings and in the context of HIV prevention. This study assessed the validity and reliability of HIV self-testing (HIVST) acceptability scores measured using the TFA among adolescent girls and young women (AGYW) in Uganda.Methods We enrolled 377 AGYW aged 15–24 years in a cross sectional study and a structured questionnaire was used to collect data. The primary outcome was acceptability of HIVST defined as willingness to accept HIVST services if offered – measured using the seven constructs of the TFA where each was assessed with one 5-level Likert item question. Construct validation of TFA was examined using the exploratory factor analysis (EFA), confirmatory factor analysis (CFA) – through structural equation modelling (SEM), and convergent and divergent validity, while reliability was assessed with the Cronbach’s alpha. Factor extraction was guided by the scree plot, and factor rotation was performed using Oblimin method. Convergent and divergent validity were assessed by correlating the TFA acceptability scores with willingness to use HIVST and perceived HIV risk respectively.Results The median age of participants was 20 years (IQR: 18, 22). Although the scree plot suggested that three factors should be extracted, the rotated solution of the EFA yielded a single dominant factor, with all seven items loading strongly onto one latent factor. In the CFA, both SEM and generalized SEM showed consistent direction and strength of association between the TFA domains and the underlying acceptability construct despite the poor SEM global fit indices (RMSEA = 1.224, CFI = 0.173, TLI= -0.103). The correlation between acceptability scores and willingness to use HIVST, and perceived HIV risk was 0.7 and 0.1 respectively. The overall Cronbach’s alpha for the questionnaire items was 0.889.Conclusions This study provides empirical evidence supporting the construct validity and reliability of the TFA-measured acceptability scores among AGYW. All seven constructs demonstrated strong loadings on a single latent factor, indicating that the TFA operates as a coherent and unidimensional tool for measuring overall acceptability.