Phospholipidosis (PLD) is an accumulation of phospholipids in lysosome-derived multilamellar vesicles. More than 50 commercial drugs are known to cause PLD. In vitro screening assays were developed in HepG2 cells, rat primary hepatocytes, and rhesus monkey hepatocytes using the fluorescent-labeled phospholipid probe N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE) or Nile Red lipid stain. The assays were qualified using amiodarone and fluoxetine as positive controls and precocene and valproic acid as negative controls. NBD-PE accumulation and Nile Red staining were first measured using fluorescence microscopy with morphometric analysis, and the throughput of the NBD-PE assay in HepG2 cells was increased by measuring fluorescence with a multiwell spectrofluorometer. The PLD potential values obtained for the tested compounds from the morphometric analysis were similar to the values obtained from the spectrofluorometer, suggesting the plate reader assay was effective at measuring the induction of NBD-PE accumulation. Fifteen commercial compounds were evaluated using the NBD-PE assay in HepG2 cells, rat primary hepatocytes, and rhesus monkey hepatocytes. The relative NBD-PE accumulation and PLD potentials of the evaluated compounds were similar and comparable to the values observed from other in vitro PLD assays referenced in the literature using different probes and cell lines. NBD-PE accumulations observed in rat hepatocytes after drug treatments were similar to those in HepG2 cells. NBD-PE accumulation potential observed in rhesus monkey hepatocytes after drug treatment was different for tamoxifen, perhexiline, clomipramine, and haloperidol. These agents caused potent NBD-PE accumulation in HepG2 cells, but minimal or no activity was observed in rhesus monkey hepatocytes. These data suggest that the NBD-PE spectrofluorometer assay in HepG2 cells has the speed and throughput to sensitively and quantitatively determine the PLD potential of various drug candidates. In addition, these data demonstrate the species differences in PLD potential between rat and monkey hepatocytes.