This study presents the validation process of the Questionnaire on Academic Stress in Secondary Education –QASSE-, designed to assess the wide variety of school sources and situations related to academic stress in adolescence, and their relationship with students’ physical and psychological well-being. Participants were 860 Spanish high school students (52.9% girls) with an average age of 14.62 years (SD = 1.8). Through a cross-validation process, EFA and CFA supported QASSE multifactorial structure with four first-order factors -academic overload, interaction with classmates, family pressure, and future-oriented perspective- and a second-order factor of academic stress, showing a significant and intense relationship with adolescents’ psychological and physical well-being. Results also highlight the effects of the gender*educational level interaction on the students’ stress, with girls showing higher levels of stress in the transition courses between educational phases (sophomore and junior years). The QASSE demonstrates good validity and reliability, showing potential for both research and educational application. The results show the high impact of the QASSE dimensions on psychological and physical well-being in adolescence, highlighting its special usefulness for designing and adjusting educational prevention and intervention actions in this area to the students’ specific characteristics and needs