Background & Objective: Due to individual and organizational reasons, the retention of faculty members in medical science universities has become a critical challenge. Identifying and prioritizing the factors influencing faculty retention is essential. This study aimed to develop and validate a questionnaire designed to assess faculty retention at medical science universities. Materials & Methods: This psychometric study, employing a multi-phase instrument development approach, was conducted in 2023 in Iran. The study consisted of two main phases: item generation and psychometric evaluation. A 25-item preliminary questionnaire was developed based on qualitative interviews with faculty members from several medical sciences universities. In the psychometric phase, face and content validity were assessed using expert judgment, and the Content Validity Index and Content Validity Ratio were calculated based on Lawshe's method. Construct validity was examined using exploratory factor analysis and confirmatory factor analysis. Reliability was evaluated through internal consistency (Cronbach's alpha) and stability (test-retest method using Spearman-Brown coefficient). The sample consisted of 351 faculty members selected through convenience sampling. Results: The final questionnaire included 21 items across three domains: individual factors, institutional factors, and socio-political factors. The CVI and CVR values were 0.91 and 0.84, respectively. EFA revealed a three-factor structure that explained 64% of the total variance. The results of the Confirmatory Factor Analysis (CFA) indicated that the fit indices for the questionnaire's three-factor structure were appropriate. Cronbach's alpha for the total scale was 0.80, and domain-specific alphas ranged from 0.78 to 0.82. The stability assessment indicated acceptable reliability (Spearman-Brown coefficient = 0.78). Conclusions: The developed questionnaire demonstrates acceptable validity and reliability for assessing factors related to faculty retention in medical science universities. Further validation in diverse academic contexts is recommended to enhance generalizability.