A rapid method was developed to analyze δ-bilirubin (Bδ), diconjugated bilirubin (DCB), monoconjugated bilirubin (MCB), and unconjugated bilirubin (Bu) by direct injection of sera using high-performance liquid chromatography (HPLC) with an internal-surface reversed-phase silica support (ISRP) column. Sharp bilirubin peaks were obtained using a simple mobile phase of acetonitrile: 0.5 M Tris-HCI buffer (20:80, v/v, pH 7.2). A variable-wavelength detector set at 450 nm, 0.01 absorbance unit full scale (AUFS), and a recorder set at 4 mm/min were used for detection. Peaks for Bδ, DCB, MCB and Bu appeared at 4.4, 6.4, 9.2 and 14.5 min, respectively, in human serum from subject with obstructive jaundice which was used as a bilirubin standard throughout this experiment. The mean recovery rate after direct addition of Bu in swine serum was 91.9% and that of DCB was 95.9%. When sera from icteric cattle, pigs and horses were analyzed using the direct injection technique, four bilirubin peaks were obtained and there was reliable correlation between the sum of the bilirubin peak heights observed on HPLC and the total bilirubin value measured by a standard reference procedure.