Our objective was to analyze the relationship between transcutaneous bilirubin (TcB) measured on an unexposed area of skin and total serum bilirubin (TSB) in preterm infants before, during, and after phototherapy (PT). For this purpose paired TSB and TcB levels were measured daily during the first ten days after birth in preterm infants of less than 32 weeks' gestation. TcB was measured with a Dräger Jaundice Meter JM-103 on the covered hipbone. Agreement between TSB and TcB levels was assessed before, during, and after PT. True negative and corresponding false negative percentages were calculated using different TcB cut-off levels. Data are presented as mean (±SD). We obtained 856 paired TcB and TSB levels in 109 preterm infants (66 boys, gestational age 29.4 ± 1.6 weeks and birth weight 1282 g ± 316 g). We found that the difference between TSB and TcB before PT was significantly lower, 44 (±36) μmol/L, than the difference during and after PT, 61 (±29) μmol/L and 63 (±25) μmol/L, respectively; P < 0.01. Blood sampling could be reduced by 42%, with 2% false negatives, when 50 μmol/L was added to the TcB level at 70% of the PT threshold. Our conclusion is that phototherapy enhances underestimation of TSB by TcB in preterms, even if measured on unexposed skin. The use of specific TcB cut-off levels substantially reduces the need for TSB measurements.