This study reports the evaluation of four urinary biomarkers of renal toxicity, α-glutathione-S-transferase (α-GST), μ-GST, clusterin, and renal papillary antigen-1 (RPA-1), in male Sprague-Dawley and Han-Wistar rats given cisplatin, gentamicin, or N-phenylanthranilic acid (NPAA). Kidney injury was diagnosed histopathologically, according to site/nature of renal injury, and graded for severity. The area under the receiver operating characteristic (ROC) curve was used to compare the diagnostic accuracy of each exploratory renal biomarker with traditional indicators of renal function and injury (blood urea nitrogen [BUN], serum creatinine [sCr] as well as urinary N-acetyl-β-D-glucosaminidase [NAG] and protein). These analyses showed that increased urinary α-GST was superior to BUN, sCr, and NAG for diagnosis of proximal tubular (PT) degeneration/necrosis. Paradoxically, urinary α-GST was decreased in the presence of collecting duct (CD) injury without PT injury (NPAA administration). RPA-1 demonstrated high specificity for CD injury, superior to all of the reference biomarkers. The clusterin response correlated well with tubular injury, whatever the location, particularly when regeneration was present (superior to all of the reference markers for cortical tubular regeneration). There was no conclusive evidence for the diagnostic utility of μ-GST. The data were submitted for qualification review by the European Medicines Agency and the US Food and Drug Administration. Both agencies concluded that the data justified the qualification of RPA-1 and increased the level of evidence for, and clarified the context of use of, the previously qualified clusterin for use in male rats. These biomarkers can be used in conjunction with traditional clinical chemistry markers and histopathology in Good Laboratory Practice rodent toxicology studies used to support renal safety studies in clinical trials. Qualification of α-GST must await further explanation of the differences in response to PT and CD injury.