Background: The heel stick is now the most common way to draw a neonate's blood for neonate screening tests and is usually done within 3 to 7 days after birth. Simulation-based training serves as a link between classroom learning and clinical practice. It aids nursing competency before working with neonates in a real-life environment, hence improving the quality of care and ensuring neonates' safety. This study aimed to evaluate the effect of simulation-based training on nurses' performance in procedural pain management of neonates' heel sampling. Method: To achieve the aim of this study, a quasi-experimental design (one group pre/ post-test design) was used. Setting: The study was conducted in the Neonatal Intensive Care Unit affiliated with Mansoura University Hospital. Subjects: A convenience sample composed of 50 neonatal nurses regardless of their age, gender, qualifications, and experiences, who were working at the previously mentioned settings. Also, all available neonates comprised 50 undergoing heel stick puncture regardless of their gestational age, gender, and birth weight during the research period. Tools for data collection: Tool I: A Structured Interviewing Questionnaire, Tool II: Observational Checklists, Tool III. Neonatal Infant Pain Scale was used to collect data. Results: There was a highly statistically significant difference between the studied nurses' knowledge and practice. The study result revealed that more than two-thirds of the studied nurses had a poor level of knowledge, and more than half of the nurses had an incompetent level of practice about neonates' heel sampling before implementing the simulation-based training. The vast majority of the studied nurses had a good level of knowledge and most of them had a competent level of practice after implementing simulation-based training. There was a highly statistically significant difference and improvement in nurses' performance after simulation-based training than pre-training. Conclusion: The present study concluded that simulation-based training had a positive effect on improving nurses' performance regarding procedural pain management of neonates' heel sampling. Recommendations: The study recommended that simulation- based training should be integrated as an effective method in nurses' training about neonates' heel sampling.