Abstract: Objective To understand the characteristics of serum bile acid spectrum, and to explore the value of bile acid spectrum in predicting breast milk jaundice. Methods Total 320 normal neonatus delivered in our hospital were selected. According to the hourly bilirubin chart of newborns, newborns whose bilirubin level was below 75% were collected at 72 hours after birth. The blood samples were sealed and refrigerated for inspection and follow-up. According to the follow-up results, the neonatus were divided into breast milk jaundice group(n=36) and normal neonatal group(n=48). The concentrations of 15 kinds of bile acids were measured by ultra high performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS). Results There were some differences in blood bile acid profiles between the two groups. The difference of glycine cholic acid [(0.41±0.31) nmol/L and(0.58±0.39) nmol/L], the ratio of glycine cholic acid to ursodeoxycholic acid(0.11±0.19 and 0.30±0.51) and the ratio of ursodeoxycholic acid to total ursodeoxychol(0.28±0.23 and 0.19±0.14) were statistically significant(all P<0.05). There was no significant difference in the levels of cholic acid, deoxycholic acid, lithocholic acid, anthropodesoxycholic acid, ursodeoxycholic acid, glycocholic acid, glycodesoxycholic acid, glycochenodeoxycholic acid, glycine ursodeoxycholic acid, cholaic acid, taurodeoxycholic acid, taurolithocholic acid, taurine goose deoxycholic acid, tauroursodeoxycholic acid, total bile acid and cholic acid/anthropodesoxycholic acid between the two groups(all P>0.05). Conclusion UPLC-MS/MS method can sensitively detect the changes of blood bile acid spectrum in neonatal breast milk jaundice before the occurrence of hyperbilirubinemia, which is helpful to predict hyperbilirubinemia and its etiology in the early stage.