A new and simple colorimetric method for human serum lipase [EC 3. 1. 1. 3] assay has been developed, using 2, 3-dimercaptopropan-l-ol tributyroate as a substrate, 5, 5'-dithiobis(2-nitro-benzoic acid) as a chromogenic reagent, phenylmethylsulfonyl fluoride as an inhibitor of serum esterases, and sodium dodecylsulfate as a lipase activator. The method requires only 50 μl×2 of serum sample and a reaction time of less than 30min. The method is reproducible and sensitive enough to measure low levels of lipase activity in normal and abnormal sera. The gel filtration of serum samples on a Sephadex G-200 column gave one peak of lipase activity, when measured by the present method, and the molecular weight of the enzyme was identical with that of lipase of human pancreatic origin, confirming the specificity of this new method for the serum lipase.