Phospholipidosis and steatosis are two toxic effects, which course with overaccumulation of different classes of lipids in the liver. MS-based lipidomics has become a powerful tool for the comprehensive determination of lipids. LC-MS lipid profiling of HepG2 cells is proposed as an in vitro assay to study and anticipate phospholipidosis and steatosis. Cells with and without pre-incubation with a mixture of free fatty acids (FFA) (i.e., oleic and palmitic) were exposed to a set of well-known steatogenic and phospholipidogenic compounds. The use of FFA pre-loading accelerated the accumulation of phospholipids thus leading to a better discrimination of phospholipidosis, and magnified the lipidomic alterations induced by steatogenic drugs. Phospholipidosis was characterized by increased levels of phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines and phosphatidylinositols, while steatosis induced alterations in FA oxidation and triacylglyceride (TG) synthesis pathways (with changes in the levels of FFA, acylcarnitines, monoacylglycerides, diacylglycerides and TG). Interestingly, palmitic and oleic acids incorporation into lipids differed. A characteristic pattern was observed in the fold of change of particular TG species in the case of steatosis (TG(54:3) > TG(52:2) > TG(50:1) > TG(48:0)). Based on the levels of those lipids containing only palmitic and or oleic acid moieties a PLSDA model was built, which showed good discrimination among non-toxic, phospholipidogenic and steatogenic compounds. In conclusion it has been shown that the use of FFA pre-incubation together with intracellular LC-MS-based lipid profiling could be a useful approach to identify the potential of drug candidates to induce phospholipidosis and/or steatosis. This article is protected by copyright. All rights reserved.