Objective: We studied the reliability and validity of the COMFORTneo scale, designed to measure neonatal prolonged pain.Study design: This prospective observational study evaluated four clinimetric properties of the COMFORTneo scale from NICU nurses’ assessments of neonates’ pain. Intra-rater reliability was determined from three video fragments at two time points. Inter-rater reliability and construct validity were determined in five neonates per nurse with the COMFORTneo and numeric rating scales (NRS) for pain and distress. Pain scores using N-PASS were correlated with COMFORTneo scores to further evaluate construct validity.Result: Intra-rater reliability: Twenty-two nurses assessed pain twice with an intraclass correlation coefficient (ICC) of 0.70. Inter-rater reliability: The ICC for 310 COMFORTneo scores together with 62 nurses was 0.93. Construct validity: Correlation between COMFORTneo and NRS pain, distress, and N-PASS was 0.34, 0.72, and 0.70, respectively.Conclusion: The COMFORTneo can be used to reliably and validly assess pain in NICU patients.