Introduction: Industry-specific safety climate scales that measure safety status have been published, however, nothing specific to biological laboratories has ever been established.Objective: This study aimed to develop and validate a biosafety climate (BSCL) scale unique for research professionals (RPs) and biosafety professionals (BPs) at teaching and research biological laboratories affiliated to public universities in the United States. Methods: BSCL scale was developed from literature review. In study 1, 15-item biosafety climate (BSCL-15) scale with 15 items and 5 factors was pretested with n = 9 RPs and n = 7 BPs to perform reliability, content, and face validity analyses. In study 2, revised 17-item biosafety climate (BSCL-17) scale with 17 items and 5 factors was pilot tested with n = 91 RPs and n = 88 BPs. Correlation tests, Kaiser-Mayer-Olkin, Bartlett's test of sphericity, Cronbach's alpha, and exploratory factor analysis (EFA) were conducted to validate the BSCL-17 scale. Results: EFA resulted in a 3-factor 17-item BSCL scale for both RPs and BPs. Internal consistency of the scale was > 0.8 for the BSCL scale and the underlying three factors, indicating high reliability. The factors identified for RPs are 1) management priority, communication and participation, 2) group norms, and 3) supervisor commitment. The factors identified for BPs are 1) management priority and communication, 2) group norms and participation, and 3) supervisor commitment. Discussion: A valid and reliable BSCL scale to measure safety climate and quantify safety culture in biological laboratories has been presented. It can be used as a key performance indicator and aid in targeted interventions as part of process improvement of biological safety programs.