Niemann–Pick disease type C (NPC) is an autosomal recessive disease caused by a functional deficiency of cholesterol-transporting proteins in lysosomes, and exhibits various clinical symp-toms. Since mitochondrial dysfunction in NPC has recently been reported, cholesterol catabolism to steroid hormones may consequently be impaired. In this study, we developed a comprehensive steroid hormone analysis method using liquid chromatography/tandem mass spectrometry (LC– MS/MS) and applied it to analyze changes in steroid hormone concentrations in NPC model cells. We investigated the analytical conditions for simultaneous LC–MS/MS analysis, which could be readily separated from each other and showed good reproducibility. The NPC phenotype was verified as an NPC model with mitochondrial abnormalities using filipin staining and organelle morphology obser-vations. Steroid hormones in the cell suspension and cell culture medium were also analyzed. Steroid hormone analysis indicated that the levels of six steroid hormones were significantly decreased in the NPC model cell and culture medium compared to those in the wild-type cell and culture medium. These results indicate that some steroid hormones change during NPC pathophysiology and this change is accompanied by mitochondrial abnormalities.