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For the quantitative determination of direct and total bilirubin in human serum. Bilirubin is a metabolite of the heme portion of heme proteins, mainly hemoglobin. Normally, bilirubin is excreted into the intestine and bile from the liver. The site of the catabolism of hemoglobin is the reticuloendothelial system (RES). Bilirubin is then released into the bloodstream where it binds tightly to albumin and is transported to the liver. Upon uptake by the liver, bilirubin is conjugated with glucuronic acid to form bilirubin mono and diglucuronide, which are water-soluble metabolites. The metabolites will react with aqueous diazo reagent and are commonly referred to as “direct bilirubin”. Elevation of total serum bilirubin may occur due to (1) excessive hemolysis or destruction of the red blood cells (e.g. hemolytic disease of the newborn), (2) liver diseases (e.g. hepatitis and cirrhosis), (3) obstruction of the biliary tract (e.g.. gallstones). There is information in the literature indicating elevated levels of direct bilirubin in patients with liver or biliary tract disease, even though total bilirubin levels are normal. Therefore, the greatest diagnostic value of direct bilirubin assays stem from their ability to indicate occult liver disease.