As neonates are submitted to pain, assessing the pain is crucial in effective pain control. The Premature Infant Pain Profile, an acute measurement tool combining physiological, behavioural and contextual indicators, was translated into Norwegian and tested clinically. The purpose was to establish construct validity, interrater reliability and internal consistency. In addition, the effect of sucrose as pain analgesia was tested in neonates ≥ 36 weeks of gestational age. In a known-groups comparisons design with repeated measures, 111 consecutive neonates, preterm and term, were all observed at baseline, non-pain and pain event. Neonates in the neonatal unit received sucrose at pain event. A significant interaction effect of gestational age and events was found in the sucrose neonates. A significant interaction effect was detected from sucrose and event type for neonates from 36 weeks. The internal consistency of the six-item score was acceptable. A correlation coefficient of 0.89–0.97 was obtained for interrater reliability. The Norwegian version of the Premature Infant Pain Profile seems to be a reliable and valid instrument for pain assessment in neonates.