Background: Pain is a major source of neonatal distress. Non-pharmacological pain management refers to use of drug-free to relieve pain. That is effective, low risks to neonates and low operating cost. Aim: This study aimed to assess nurses' performance regarding non-pharmacological pain management of neonates in intensive care unit. Design: A descriptive design was used. Setting: This study carried out at Neonatal Intensive Care Units at Fayoum General Hospital affiliated to Ministry of health and Fayoum Health Assurance Hospital affiliated to Health Assurance Organization. Sample: A purposive sample consisted of 100 nurses who providing care for neonates. Tools: Two tools used for data collection, Tool (1) A structured interview questionnaire consisted of two parts: Part (1) Characteristics of studied nurses. Part (2) Knowledge of studied nurses regarding neonatal pain and non-pharmacological management. Tool (2) Observational checklist to assess practice of studied nurses regarding non-pharmacological pain management. Results: Revealed that, the mean age of the studied nurses 33.3 ± 3.9 years and 52% of the studied nurses had poor level of knowledge. As well, 82% of them had incompetent practice regarding neonatal pain and non-pharmacological management methods. Also, there were highly significant positive correlation between total knowledge of studied nurses and their reported total practice regarding neonatal pain and non-pharmacological management methods. Conclusion: The present study concluded that, more than half had poor level of knowledge and the majority had incompetent practice regarding neonatal pain and non-pharmacological pain management methods. Recommendations: Continuous training program for neonatal nurses about non-pharmacological pain management