Background: Chemistry laboratories of universities are dangerous. However, there is no appropriate instrument to assess staffs' awareness. The present study aims at developing a standardized instrument for awareness assessment of laboratory personnel on safe work.Methods: The present study is a kind of developing instrument methodology. The study was implemented in two phases: (I) generating items based on the review of relevant literature and interviews, and (II) assessment of the content validity according to seven occupational health and laboratory sciences experts. The validity of the tool was determined based on content validity index (CVI) and content validity ratio (CVR).Results: In the item generation phase, 53 items were developed in four areas including storage of chemical substances, usage of chemical substances, personal protection equipment, and general observations. The value of CVI was in the range of 0.75-0.91, while the CVR was in the range of 0.4 to 1. Based on these values, some items were omitted and, finally, the content validity for the 40-item questionnaire was established.Conclusion: Since the content validity of this questionnaire was established, it can be used in future studies.