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Non-alcoholic steatohepatitis (NASH) is a condition characterized by inflammation and hepatic injury/fibrosis caused by the accumulation of ectopic fats in the liver. Recent advances in lipidomics have allowed the identification and characterization of lipid species and have revealed signature patterns of various diseases. Here, we describe a lipidomics workflow to assess the lipid profiles of liver homogenates taken from a NASH mouse model. The protocol described below was used to extract and analyze the metabolites from the livers of mice with NASH by liquid chromatography–mass spectrometry (LC-MS); however, it can be applied to other tissue homogenate samples. Using this method, over 1,000 species of lipids from five classes can be analyzed in a single run on the LC-MS. Also, partial elucidation of the identity of neutral lipid (triacylglycerides and diacylglycerides) aliphatic chains can be performed with this simple LC-MS setup.Key features• Over 1,000 lipid species (sphingolipids, cholesteryl esters, neutral lipids, phospholipids, fatty acids) are analyzed in one run.• Analysis of liver lipids in non-alcoholic steatohepatitis (NASH) mouse model.• Normal-phase chromatography coupled to a triple quadrupole mass spectrometer.Graphical overviewSchematic procedure for the homogenization and extraction of mouse liver tissue in preparation for LC-MS analysis (Created with BioRender.com)