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The Premature Infant Pain Profile (PIPP) is a 7-indicator composite measure developed to assess acute pain in preterm and term neonates. It has been validated in studies using synchronized videotaping of infants undergoing procedures. The purpose of this study was to establish (a) construct validity of the PIPP and (b) inter- and intrarater reliability of the PIPP prospectively in the clinical environment.A randomized, crossover design was used.The study was conducted in a Level III outborn neonatal intensive care unit.A convenience sample of 43 neonates, stratified by gestational age, was studied.Each infant experienced three separate, randomly ordered events: baseline, a painful event, and a nonpain event. Infants were videotaped and scored at the bedside using the PIPP by the nurse caring for the infant and the clinical nurse specialist who had expertise in infant pain. The videotapes were later reviewed by two additional experts; one in real time and one using a second-to-second stop frame technique.Repeated-measures analysis of the main effects and interactions yielded a statistically significant main effect for event (pain, nonpain, baseline), thus differentiating pain from nonpain and baseline events (F = 48, p = 0.0001) and establishing construct validity. Interrater reliability analysis of individual event scores of the PIPP yielded reliability coefficients of 0.93-0.96. Intrarater reliability coefficients analysis for individual events were equally high at 0.94-0.98.This study demonstrates that the PIPP is a pain measure with good construct validity and excellent inter- and intrarater reliability for the assessment of procedural pain of preterm and term infants in clinical settings.