Biliary Atresia (BA), a result from inflammatory destruction of the intrahepatic and extrahepatic bile ducts, is a severe hepatobiliary disorder unique to infancy. Early diagnosis and Kasai operation greatly improve the outcome of BA patients, which encourages the development of early screening methods. Using HPLC coupled tandem mass spectrometry, we detected primary bile acids content in dried blood spots obtained from 8 BA infants, 17 neonatal jaundice and 292 comparison infants at 3-4 days of life. Taurocholate (TC) was significantly elevated in biliary atresia infants (0.98 ± 0.62 µmol/L) compared to neonatal jaundice (0.47 ± 0.30 µmol/L) and comparison infants (0.43 ± 0.40 µmol/L), with p=0.0231 and p=0.0016 respectively. The area under receiver operating characteristic (ROC) curve for TC to discriminate BA and comparison infants was 0.82 (95% confidence interval: 0.72-0.92). A cutoff of 0.63 µmol/L produced a sensitivity of 79.1% and specificity of 62.5%. The concentrations of total bile acids were also raised significantly in BA compared to comparison infants (6.62 ± 3.89 µmol/L vs 3.81 ± 3.06 µmol/L, p=0.0162), with the area under ROC curve of 0.75 (95% confidence interval: 0.61-0.89). No significant difference was found between the bile acids of neonatal jaundice and that of comparison infants. The early increase of bile acids indicates the presentation of BA in the immediate newborn period and the possibility of TC as newborn screening marker.