Background: Neonatal pain management is a professional practice, education and research opportunity that nurses need to explore, evaluate and refine continuously within the rapidly changing technological environment of the Neonatal Intensive Care Units (NICUs). Aim of the study: This study aimed at assess nurses' performance regarding pain management of neonates undergoing heel stick puncture. Design: A descriptive design was used to conduct this study. Setting: This study was conducted at the Neonatal Intensive Care Unit in El-Fayoum General Hospital. A convenience sample composed of 70 nurses who were working at the previously mentioned study settings and 70 neonates during the study period. Tools: Pre-designed Questionnaire Sheet, Observational Checklists, Likert Type Rating Scale. and Neonatal Infant Pain Scale. Results: It was found that two thirds of the studied nurses had good knowledge regarding heel stick puncture and pain management, most of them had incompetent practices regarding heel stick puncture and pain management of the neonates. Majority of nurses had positive attitude regarding pain management of neonates, and three fifths of the neonates had mild to moderate pain. Conclusion: Nearly more than two thirds of the studied nurses had good knowledge and most of them had incompetent practices regarding heel stick puncture and pain management of the neonates, Also the majority of the studied nurses had positive attitude regarding pain management of neonates undergoing heel stick puncture. Recommendation: Periodic assessment of nurse's performances regarding pain management of neonates undergoing heel stick puncture.