Abstract: An improved conjugated (direct) bilirubin assay comprises treating a sample to be assayed with a buffer having a pH of about 4 to 5 and containing iodide ions thereby decreasing the oxidation of hemoglobin to methemoglobin and preventing conjugated bilirubin from being oxidized to biliverdin. The iodide also prevents the destruction of the azopigment by the hemoglobin. The assay further comprises adding a diazo reagent to the treated sample, incubating the resulting mixture, adding ascorbic acid, alkaline tartrate and a caffeine reagent and then reading the absorbence at 600 nm. The concentration of direct bilirubin is calculated by comparison to the absorbance of unconjugated standard(s) analyzed by a method for total bilirubin. The improved assay is more accurate than prior art methods because hemoglobin interference is negligible. In a preferred embodiment the buffer is sodium acetate buffer having a pH of about 4.75 which contains 3 to 30 grams/L of potassium iodide.