Background: Despite huge advances in science, technology, and medical equipment, numerous concerns grow over healthcare workers (HCWs) since they are exposed to a wide range of biological hazards due to the nature of their work. Objectives: The current study aimed at developing and validating an index to assess the risk of occupational safety and health (OSH) in hospitals and healthcare settings. Methods: In the current cross-sectional study, an index called the hospital occupational safety, and health risk assessment (HOSHRA) was developed and validated through the face and content validity as well as internal consistency and inter-rater reliability. Ultimately, the developed index was utilized to assess OSH risks in 36 teaching hospitals affiliated to Shiraz University of Medical Sciences, Shiraz, Iran. Results: The mean score of the HOSHRA index among the selected hospitals was 71.53 ± 5.93, indicating that the OSH risks were at a moderate level. According to the HOSHRA action levels, OSH risk level for ergonomic hazards was high, but it was moderate for physical, chemical, ergonomic, and psychological ones. In terms of physical hazard subcategories, the risk of electrical hazard, as well as fire and explosion, was high, but it was moderate for fall and slip, and radiation. Conclusions: The developed observation-based method showed an acceptable content validity and reliability for OSH risk assessment of hospitals. The HOSHRA index could also be used for hospitals as an applicable measure to improve their OSH.