Gender bias is a pervasive problem with significant negative outcomes for women leaders and organizations. Bias manifests in a multiplicity of forms, ranging from subtle to overt. To date, no instrument exists to measure women leaders' perceptions of gender bias. This study presents a comprehensive measure of how women leaders perceive and experience gender bias. Drawing from a national sample of female higher education executives (n = 488), faith‐based organization leaders (n = 298), physicians (n = 293), and attorneys (n = 527), the present research developed and validated the Gender Bias Scale for Women Leaders. Building upon a previously established and expansive cross‐level conceptual framework to develop the initial item pool, results from both exploratory and confirmatory factor analysis supported a factor structure consisting of six higher‐order and 15 lower‐order factors. Our results show that several factors of the Gender Bias Scale correlated with workplace outcomes of turnover intention and satisfaction. Human resource development (HRD) professionals may use the Gender Bias Scale to identify the specific types of bias prevalent for women leaders in their organizations and apply interventions accordingly. Implications for women leaders and HRD professionals and recommendations for future research are discussed.