Objective To evaluate the effectiveness of cerebral regional oxygen saturation (crSO2) values, measured using near-infrared spectroscopy (NIRS), in assessing pain associated with the peripherally inserted central catheter (PICC) in premature infants. Methods NIRS was used to measure the crSO2 levels of 48 premature infants with gestational age (GA) of < 32 weeks or a birth weight of < 1500 g. Premature infant pain profile (PIPP) scores, vital signs, transcutaneous oxygen tension (TcpO2), transcutaneous carbon dioxide tension (TcpCO2), and crSO2 values were monitored. One-way repeated measure analysis of variance was used to compare heart rate (HR), respiratory rate (RR), blood pressure (BP), peripheral oxygen saturation (SpO2), TcpO2, TcpCO2, and crSO2 values before (Time 1), during (Time 2), and after (Time 3) PICC insertion. The correlation between the PIPP scores at Time 2 and the fluctuations (values detected at Time 2 minus those at Time 1) of SpO2, TcpO2, and crSO2 were also analyzed. Results The PIPP score at Time 2 was significantly higher than those at Times 1 and 3. HR, RR, and BP values increased (p < .05), and SpO2 and crSO2 levels decreased at Time 2 (p < .05) compared with those at Time 1. Stratified analysis based on GA revealed significant differences in HR, RR, and crSO2 values between Times 1 and 2 in infants with a GA of ≥ 32 weeks. In infants with a GA < 32 weeks, significant differences were observed in HR, RR, SpO2, BP, and crSO2 values between Times 1 and 2. The fluctuation of the crSO2 level was strongly correlated with the PIPP score at Time 2 (r = −0.829, p < .001). A weak correlation was observed between the PIPP score at Time 2 and TcpO2 level fluctuation (r = 0.375, p = .009). No correlation was observed between the PIPP score at Time 2 and SpO2 level fluctuation (r = 0.242, p = .097). Conclusion The fluctuation of crSO2 levels strongly correlates with PICC procedural pain. Hence, crSO2 levels measured using NIRS may be used as an indicator for pain assessment in premature infants.