Bile acid concentrations are elevated in the blood of neonates with cholestatic hepatobiliary disorders providing a possible means of screening for treatable conditions including biliary atresia. A method is described for the determination of concentrations of conjugated bile acids in dried blood spots using electrospray ionization mass spectrometry. Bile acids were eluted from the blood spots using methanol containing, as internal standards, the taurine and glycine conjugates of D4-chenodeoxycholic acid and D4-cholic acid. The samples were then reconstituted in acetonitrile/water and injected by autosampler into the electrospray source operating in negative ion mode. Optimal conditions were determined for both single quadrupole and tandem mass spectrometry analysis. Blood spot bile acid profiles were studied in two groups of infants (<1 y), a cholestatic group(conjugated bilirubin >25 μmol/L; n = 49), and a control group(n = 96). The best discrimination between the two groups was provided by measurements of taurodihydroxycholanoates (normal <5 μmol/L; cholestatic group 18-94 μmol/L) and glycodihydroxycholanoates (normal <5μmol/L; cholestatic group 11-66 μmol/L). The method can also be adapted to detect unusual bile acids which are diagnostic of inborn errors of bile acid synthesis and peroxisomal disorders. The method is fast, reliable, reproducible, and relatively cheap; however, much more work is required to determine whether it can be used for mass screening for cholestasis. It will be necessary to show that measurement of bile acid concentrations in blood spots obtained at 7-10 d can be used to detect infants who currently present with jaundice, pale stools, and dark urine during the first 6 mo of life.