Background: Transcutaneous bilirubin is used as a screening tool before starting phototherapy in preterm newborn. However, there is conflicting evidence regarding its utility after starting phototherapy. There is also doubt regarding the best site of measurement. Objective: The objective was to analyze the association between transcutaneous bilirubin and serum bilirubin in Indian preterm newborns at different sites before, during, and after phototherapy. Methods: This prospective observational study was conducted in a tertiary care neonatal unit of eastern India from May 2019 to April 2020. Participants: The study participants were 90 preterm newborns admitted for phototherapy within the first 7 days of life. Intervention: Transcutaneous bilirubin was assessed by Draeger JM-105 at the covered areas of the sternum and forehead and serum bilirubin was assessed by the Diazo method within 30 min of each other. Bilirubin assessment was done before starting phototherapy and then repeated every 12 h till 24 h postphototherapy. Outcome: To determine the correlation and agreement between serum bilirubin and transcutaneous bilirubin in preterm newborns before, during and after phototherapy. Results: Median gestational age and mean birth weight of the enrolled infants were 33.5 weeks and 1920 g, respectively. Transcutaneous bilirubin from sternum demonstrated good correlation with serum bilirubin before (r = 0.856), during (r = 0.723 at 24 h), and after phototherapy (r = 0.801 at 24 h postphototherapy). Measurements from the forehead showed better correlation with serum bilirubin compared to the sternum (r = 0.865 before, 0.732 during, 0.814 after phototherapy). At both sites, correlation worsened with phototherapy but improved gradually in the postphototherapy period. Bland–Altman analysis revealed wide upper limit of agreement between transcutaneous bilirubin and serum bilirubin at both sites and all points of time, suggesting a transcutaneous device significantly overestimated serum bilirubin. Conclusion: There was good correlation but poor agreement between transcutaneous bilirubin and serum bilirubin in Indian preterm newborns irrespective of timing and site of measurement.