Background Various conjugated cholesterol metabolites are excreted in urine of the patients with metabolic abnormalities and hepatobiliary diseases. We aimed to examine the usefulness of precursor ion scan and neutral loss scan for the characterization of conjugated cholesterol metabolites in urine. Methods A mixture of authentic standards of conjugated cholesterol metabolites was used for investigating the performance of the present method. The urine of patients with Niemann–Pick diseases type C and 3β-hydroxysteroid dehydrogenase deficiency were analysed by precursor ion scan of m/z 97, 74, and 124. Results A precursor ion scan of m/z 97 was effective for identifying conjugates with ester sulphates on hydroxyl groups whereas ester sulphates on phenolic alcohols were signalled by a neutral loss scan of 80 Da. Monosaccharide-conjugated cholesterol metabolites were signalled by a precursor ion scan of m/z 113. Although precursor ion scan of m/z 74 and 124 was effective for finding glycine- and taurine-conjugated metabolites, high intensity of product ions ( m/z 74 and 124) disturbed measurement of other multiply conjugated metabolites. The urine samples contained many conjugated cholesterol metabolites, and there were several disease-specific intense peaks. We found several unknown intense peaks with three known peaks in urine of the Niemann–Pick type C patient. In the patient with 3β-hydroxysteroid dehydrogenase deficiency, intense peaks that were tentatively identified as 5-cholenoic acid sulphates and their glycine and taurine conjugates were present. Conclusion The method should lead to the discovery of new urinary biomarkers for these disturbances of cholesterol catabolism and transport.