Non-alcoholic fatty liver disease (NAFLD) is prevalent (overall occurrence of~ 20%) in the US adult population and is very commonly associated with obesity and diabetes (occurs in~ 60% of diabetic patients). NAFLD presents as increased number and size of lipid droplets within the hepatocytes, and, if untreated, can progress to the inflammatory disease steatohepatitis and liver dysfunction. This probe report discloses the identification and optimization of two highly potent nanomolar, non-cytotoxic first-in-class small molecule chemical inhibitors of hepatic lipid droplet formation in murine AML-12 cells, that represent distinct chemical scaffolds: ML261 (IC 50= 69.7 nM), a thienopyrrole-5-caboxamide and ML262 (IC 50= 6.4 nM), a phenylthiophene-2-carboxamide. A novel high-content imaged based phenotypic assay that uses automated image processing to quantitate the inhibitory effect of compounds on the number and size of lipid droplets formed upon treatment of AML-12 murine hepatocyte cell cultures with oleic acid, after fixing and staining them with a lipophilic BODIPY due. These inhibitors appear to work downstream of lipid transporters,... These inhibitors are much more potent than triacsin C (IC 50~ 1–3 μM) in AML-12 cells (as determined from this work), which is a known inhibitor of long-chain acyl-CoA synthetase (ACSL) with potency against ACSL in Raji cell membrane fractions (~ 1 μM) or cell sonicates (2.6–8.7 μM). And as compared to more recent, cellularly active inhibitors ranging in potency from 2.5 to 100 μM. Surprisingly, these inhibitors appear to be exquisitely potent in AM12 murine hepatocytes but show little activity in the corresponding HuH7 or primary human hepatocyte cells, suggesting species-specific effects.