The Neonatal Pain, Agitation, and Sedation Scale (N-PASS) is recommended by the American Academy of Pediatrics to measure neonatal pain and sedation. However, little is known regarding its reliability and validity for diverse neonatal subpopulations. Twenty-nine studies were included in our review, demonstrating broad application of N-PASS and good or excellent reliability and validity for various neonatal subpopulations. Our systematic review found N-PASS to be valid and reliable for many but not all neonatal subpopulations. There is a lack of support for N-PASS reliability and validity for measuring prolonged pain and sedation in nonmechanically ventilated infants and for acute pain in postoperative infants in any gestational age category. Overall, N-PASS is a psychometrically sound and pragmatic instrument evaluating pain and sedation for most neonatal populations. Future research using N-PASS is encouraged to evaluate and report its validity and reliability, especially for neonatal subpopulations not included in this review.