Oxycholesterols, oxidized cholesterol metabolites, are important roles for regulator of hepatic cholesterol and stimulation of reverse cholesterol transport. These metabolites also contribute to accelerating the progression of inflammatory and fibrogenic response in the liver. Therefore, oxycholesterols are associated with a broad range of metabolic disorders such as non-alcoholic steatohepatitis (NASH). For these reasons, oxycholesterols are interesting candidates as potential biomarkers of NASH. In this study, we developed analytical method and simultaneously analyzed six OHCs (24S-, 25-, 27-OHCs and 24S-, 25-, 27-diOHCs) levels in human liver microsomes. The profiles of OHCs were also performed in various animals such as dog, rat and mouse liver microsomes by LC-ESI/MS/MS with picolinyl ester (PE) derivatization. In addition, we investigated endogenous levels of OHCs in pooled normal and NASH human liver microsomes. As results, endogenous levels of 24-OHC and 27-OHC were obtained in range of 0.531 to 0.559 ng/mL and 2.652 to 2.905 ng/mL for in normal liver microsomes, and were obtained in range of 0.625 to 0.703 ng/mL and 2.269 to 2.450 ng/mL for NASH liver microsomes, respectively. The OHCs profiling showed that 24-OHC was increased and 27-OHC was reduced in NASH liver microsomes, compared with controls (p<0.01). Therefore, we presented the potential of LC-ESI/MS/MS with PE derivatization method and applied for simultaneous quantification of six OHCs in liver microsomes. The present method is clinically suitable for diagnosis. * This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2020R1F1A1071199).