Background and Aim: Prone positioning during general anesthesia is one of the most difficult practices for the perioperative nurse. Patients in this position are vulnerable to many preventable complications. However, no studies have developed an evidenced-based tool to improve nursing practice during general anesthesia and prone positioning. This study aimed to develop and test a general anesthesia and prone position nursing checklist for use by the circulating nurse. Methods: The WHO checklist development model and evidence-based methods guided the checklist development process. A prospective pre-post study was performed between November 2020 and March 2021. We prospectively observed circulating nurses that attended to prone general anesthesia during posterior lumbar spine surgery for three months before and after the introduction of the general anesthesia and prone position nursing risk checklist. The main outcomes were successful delivery of essential prone positional nursing practices during each surgery and the nurse's opinion of the checklist’s efficacy and utility. Results: A general anesthesia and prone position nursing checklist comprised of 4 pause points and 22 necessary nursing practices was developed. Seventy-two nurses participated in this study. Use of the checklist significantly increased the average performance of essential practices during each surgery from 72.72% to 95.45%. Three measures had a compliance rate of 100%. The delivery rate of 14 measures was significantly improved, 91.7% of nurses considered the checklist easy to use, and 94.4% nurses would want the checklist to be used if they underwent a prone position and general anesthesia operation. Conclusions: A general anesthesia and prone position nursing checklist was developed. The instrument was found to be an effective and positively received tool for facilitating key measures of prone positioning during nursing care, translating evidence into practice, and standardizing the nursing process.