The Sexuality Knowledge Assessment Questionnaire (SEKAQ) was designed to assess sexuality-related knowledge among adolescents and future health and education professionals, particularly within the context of school-based health education initiatives. This study developed and validated the SEKAQ, an instrument aimed at evaluating critical aspects of sexuality education, such as sexual risks, STIs, pornography, sexual diversity, gender stereotypes, sexual violence, body image issues, and societal pressures regarding virginity. Developed collaboratively with healthcare professionals and educational institutions, the SEKAQ comprises 11 items, each with multiple-choice answers and only one correct option. Psychometric evaluation was conducted using data from 454 participants, including high school, nursing, educator, and health sciences students. The SEKAQ demonstrated strong internal consistency, with a Cronbach's ⍺ of 0.84 and an average score of 7.04 (SD = 3.18). Factorial analysis confirmed the reliability and validity of the SEKAQ as a single-factor tool, identifying knowledge gaps in sexuality to guide healthcare interventions. The results showed higher knowledge among women and nursing students, while younger participants and teachers had lower scores. SEKAQ provides a valuable tool for health and education professionals to evaluate sexuality knowledge, supporting the development of sexual well-being education strategies for teenagers and youth.