Abstract: Novel reagent compositions and an assay method for the measurement of total and direct bilirubin are disclosed. The reagent compositions comprise an aqueous solution of at least one betaine selected from the group of alkyl betaine, betaine and betaine hydrate, a sulfanilamide diazonium salt, iodide ions and sulfamic acid. In the measurement of total bilirubin, alkyl betaine serves as an accelerator/solubilizer for the reaction of unconjugated bilirubin, and the alkyl betaine, betaine or betaine hydrate, together with iodide ions, minimizes or substantially eliminates interference from hemoglobin and lipemia.