Objective: to evaluate the efficacy of the Premature Infant Pain Profile (PIPP) for pain assessment in neonates and to determine the analgesic effect of fentanyl. Methods: Prospective study in 72 neonates, 60 without analgesia (control group) and 12 receiving a continuous infusion of fentanyl at 1.5 mcg/kg/h (fentanyl group). The PIPP was used for pain assessment. The heel lancing procedure was chosen as the painful event with 4 distinct components: swab, lance, squeeze and recovery. Results: PIPP scores were significantly higher during lance (median:8, range: 1-13) and squeeze (10, 2-14) than during swab (4, 0-9) and recovery (4, 0-9) (p<0.000). The fentanyl group showed significantly lower PIPP scores than the control group during lance (6, 3-12 vs 9, 1-13) and during squeeze (7, 3-12 vs 11, 2-14), p<0.000). Conclusions: PIPP scale is a useful tool for neonatal pain assessment in our clinical practice. Neonates receiving fentanyl showed lower PIPP scores after a painful event.