Niemann-Pick C disease (NPC) is a lysosomal storage disorder causing abnormal accumulation of unesterified free cholesterol in lysosomal storage organelles. High content phenotypic microscopy chemical screens in both human and hamster NPC-deficient cells have identified several compounds that partially revert the NPC phenotype. Cell biological and biochemical studies show that several of these molecules inhibit lysosomal acid lipase, the enzyme that hydrolyzes LDL-derived triacylglycerol and cholesteryl esters. The effects of reduced lysosomal acid lipase activity in lowering cholesterol accumulation in NPC mutant cells were verified by RNAi-mediated knockdown of lysosomal acid lipase in NPC1-deficient human fibroblasts. This work demonstrates the utility of phenotypic cellular screens as a means to identify molecular targets for altering a complex process such as intracellular cholesterol trafficking and metabolism.