Nonalcoholic steatohepatitis (NASH) is a disease with symptoms similar to those of alcoholic liver inflammation without alcohol intake. As an effective treatment strategy has not been established for this disease, a detailed understanding of the pathological progression mechanism is required. We focused on cholesterol metabolites, which are suspected to regulate NASH pathology, and investigated their relationship with the pathological progression in the early stages of NASH. First, the LC/MS/MS methods for bile acids and sterols were optimized and validated. Next, NASH model mice were established by feeding a choline-deficient, methionine-reduced high-fat diet, and the levels of hepatic cholesterol metabolites were measured. As a result, before the onset of NASH, desmosterol, 4β-hydroxycholesterol, campesterol, sitosterol, secondary bile acids such as taurodeoxycholic acid significantly decreased by up to 1/38 of NASH model group. Autoxidation-generated sterols significantly increased 2- to 5-fold, and various primary bile acids such as conjugated β-muricholic acids and cholic acids significantly increased 2- to 7-fold. In this study, the levels of cholesterol metabolites changed in the before the onset of NASH. These metabolic alterations involved in inflammation induction and detoxification for NASH may help the discovery of early diagnostic biomarkers in the future.