Background: Various methods are available to measure serum cholesterol concentrations. Of these, the cholesterol ester hydrolase (CEH)-cholesterol oxidase-peroxidase chromogenic method is widely used. However, this method has the disadvantage of interference by reducing substances. We developed and evaluated an endpoint assay for serum cholesterol, based on a CEH-cholesterol dehydrogenase (CDH)-ultraviolet method.Methods: Cholesterol esters are first hydrolyzed to free cholesterol by CEH. The free cholesterol is then reduced by CDH to cholest-4-ene-3-one with the simultaneous production of β-NADH from β-NAD+. At equilibrium, the CDH reaction gives incomplete conversion of cholesterol to cholest-4-ene-3-one. To overcome this disadvantage, we added hydrazine monohydrate to the reaction mixture to remove cholest-4-ene-3-one, which allowed the reaction to proceed to completion and gave stoichiometric production of β-NADH from the reaction of β-NAD+ with cholesterol.Results: We tested whether the amount of cholesterol added was equivalent to the absorbance change of NADH at 340 nm with six aqueous samples. Recoveries were 97.1–100.3%. The reaction was linear up to 20.28 mmol/L. The mean within-day (n = 20) and between-day (n = 10) imprecision (CV) was 0.29–0.43% and 0.22–0.61%, respectively. No interference by bilirubin, hemoglobin, ascorbic acid, and other reducing agents was observed. The equation obtained in comparison with the modified Abell-Levy-Brodie-Kendall method was: y = 0.992x − 0.0058 mmol/L; r = 0.997; Sy|x = 0.117 mmol/L; n = 50.Conclusion: This method is an accurate, reliable method for serum cholesterol analysis and is amenable to automation.