Introduction: The use of fluoroscopy-assisted surgical procedures has been increasing recently. The extensive use of fluoroscopy, combined with the lack of knowledge about radiation risks among operating room (OR) personnel (surgeons, anaesthetists, nurses, and radiographers), may lead to misconceptions. The perceptions and beliefs of any health professional concerning radiation protection safety (RPS) may affect their behaviour during surgery, leading to negative outcomes. The aim of this study was to construct a new, original, reliable, and valid instrument to record the beliefs, perceptions, and behaviours of health professionals during surgery, which reflects the established culture of RPS. Methods: A questionnaire was designed, consisting of 95 questions grouped into six coherent sections. The study was performed at a Greek public hospital with a high workload in terms of fluoroscopically guided surgical operations. Results: It was distributed among 136 people, and 132 completed questionnaires were collected (response rate 97%). Exploratory factor analysis was performed separately for each scale studied and Cronbach's Alpha reliability analysis was also performed. The reliability of the greater part of the new measurement tool ranged from very good to acceptable. Conclusion: The questionnaire developed in this study is a valid and reliable option for recording health professionals’ perceptions, beliefs, and behaviours concerning the RPS culture. Implications for practice: This study helps clinical radiographers to understand a possible knowledge gap about RPS and locates the specific fields that the OR personnel misunderstand. It also can support and establish local learning organisations with regular targeted staff training for health professionals, thereby leading to a generally improved RPS culture.