The liver is the systemic hub of lipid metabolism. The excessive accumulation of lipids in hepatocytes, steatosis, is a major clinical concern, whose progressive forms lead to end-stage liver disease. Currently, animal studies are the gold standard in toxicological risk assessment. Fueled by an integration of modern omics technologies, in silico models and in vitro system optimization, a new paradigm in the basis for toxicological risk assessment is emerging away from the use of animals. In recent years, in vitro assays have been developed for the early screening of the steatogenic potential of compounds. The present chapter describes an assay for the intracellular detection of lipids, a high-content screen for the distinction between steatosis and phospholipidosis, a multiparametric high-content screen for steatogenic potential and a liver X receptor reporter cell line.