A new enzymatic assay for selectively measuring conjugated bilirubin concentration in serum with use of bilirubin oxidase (BOD) has been developed. At pH 5.5 BOD can oxidize only conjugated bilirubin in the presence of reagents such as sodium fluoride and N-acetylcysteine which can decrease BOD reactivity to unconjugated bilirubin and bilirubin covalently bound to albumin (delta bilirubin). The resulting decrease in absorbance at 450 nm is linearly related to the concentration of conjugated bilirubin in serum. The BOD in this new assay was confirmed to oxidize conjugated bilirubin, and neither unconjugated nor delta bilirubin, based on both its reactivity to unconjugated bilirubin and HPLC results. This assay was found to give satisfactory results, such as in terms of the range of measurement, the reproducibility of the results, the lack of interference with coexisting substances in serum and the stability of the reagent solutions, in practical applications. The serum conjugated bilirubin concentrations determined using this assay correlate well with those determined by the HPLC analysis. This assay can be used for accurate monitoring of changes in the conjugated bilirubin concentration in patient sera. These findings suggest that the conjugated bilirubin assay is useful for fractional determination of bilirubin in icteric sera.