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Inadequate assessment of pain in premature infants is a persistent clinical problem. The objective of this research was to develop and validate a measure for assessing pain in premature infants that could be used by both clinicians and researchers.The Premature Infant Pain Profile (PIPP) was developed and validated using a prospective and retrospective design. Indicators of pain were identified from clinical experts and the literature. Indicators were retrospectively tested using four existing data sets.Infants of varying gestational ages undergoing different painful procedures from three different settings were utilized to develop and validate the measure.The largest data set (n = 124) was used to develop the PIPP. The development process included determining the factor structure of the data, developing indicators and indicator scales and establishing internal consistency. The remaining three data sets were utilized to establish beginning construct validity.The PIPP is a newly developed pain assessment measure for premature infants with beginning content and construct validity. The practicality and feasibility for using the PIPP in clinical practice will be determined in prospective research in the clinical setting.