During drug discovery, assessment of renal safety for a compound is important for further development of a candidate drug. In this study, we describe an in vitro cell-based assay capable of discerning nephrotoxicity. Three cell types, two of kidney origin and one of liver origin, were used to examine the effects of nephrotoxins. The cell types were the porcine normal kidney tubular epithelial cell line (LLC-PK1), the primary human renal proximal tubular epithelial cells (hRPTEC) and the human liver cell line (HepG2). Cytotoxicity was measured using a luciferin/luciferase assay that measures cellular ATP levels. Four known nephrotoxins, 4-aminophenol, cisplatin, cyclosporin A and paraquat, were tested in this cell-based assay to evaluate cytotoxicity on drug exposure. Kidney-derived LLC-PK1 cells and hRPTECs were found to be sensitive to selected nephrotoxins while liver-derived HepG2 cells were insensitive. Human RPTEC cells obtained from three individual donors demonstrated highly reproducible effects on drug exposure. With respect to drug discovery efforts, integration of the cell models described here are valuable for evaluation of nephrotoxic potentials during lead selection and optimization processes.