To provide a method for determining HDL cholesterol, with no need of any pretreatment such as centrifugal separation, efficient and simple in procedure, and applicable to various automatic analyzers.

This method for determining HDL cholesterol comprises as follows: a surfactant selected from polyoxyethylene alkylene phenyl ethers and polyoxyethylene alkylene phenyl ethers and an enzymatic reagent for determining the aimed cholesterol are added to serum, and the reaction level of the HDL cholesterol in lipoproteins is determined within the period of time of the preferential reaction of the HDL cholesterol with the reagent; furthermore, a substance having effect of hampering the reaction of the cholesterol in serum lipoproteins with the reagent is added.