Background: Managing neonatal jaundice effectively in preterm infants is crucial due to their increased vulnerability to bilirubin-induced neurological disorders. This study evaluates the efficacy of transcutaneous (TcB) versus serum bilirubin (TSB) measurements in this context. Methods: A total of 100 preterm neonates undergoing treatment for jaundice were assessed using both TcB and TSB measurements before, during, and after phototherapy. The study conducted paired t-tests and correlation analyses to evaluate the agreement between these two methods. Results: Before phototherapy, there was a strong positive correlation (r = 0.8319) between TcB and TSB, with a statistically significant mean difference (p = 0.0001). During phototherapy, TcB measurements were consistently lower than TSB, indicating significant discrepancies. The differences highlighted the influence of clinical interventions like phototherapy on the accuracy of TcB readings. Conclusion: The study validates transcutaneous bilirubinometry (TcB) as a non-invasive, effective alternative for jaundice monitoring in preterm infants. Despite some discrepancies with traditional serum bilirubin (TSB) measurements during phototherapy, the integration of TcB can decrease the reliance on invasive procedures. This research supports the potential of TcB to replace serum assessments, promoting a less stressful clinical experience for neonates while maintaining accuracy in therapeutic decisions. Future advancements are encouraged to enhance TcB measurement techniques to align more closely with serum bilirubin levels.