Polypeptide antibiotics, such as polymyxins and aminoglycosides, are essential for treatment of life-threatening Gram-negative infections. Acute kidney injury (AKI) attributed to treatment with these agents severely limits their clinical application. Because standard biomarkers (serum creatinine [sCRE] and blood urea nitrogen [BUN]) feature limited sensitivity, the development of novel biomarkers of AKI is important. Here, we compared the performance of standard and emerging biomarkers of AKI for the detection of nephrotoxicity caused by polymyxin B across multiple species (rat, dog and monkey). Further, we applied a biomarker-driven strategy for selection of new kidney-sparing polymyxin analogs. Polymyxin B treatment produced dose-dependent kidney injury observed as proximal tubular degeneration/regeneration and necrosis across all species. Dogs and monkeys had similar biomarker profiles that included increases of both standard (sCRE and BUN) and emerging (urinary neutrophil gelatinase-associated Lipocalin [NGAL] and urinary kidney injury molecule 1 [KIM-1]) biomarkers of AKI. In contrast, only urinary NGAL and urinary KIM-1 were sufficiently capable of detecting kidney injury in rats. Because rats provide a feasible model for screening compounds in drug development, we utilized urinary NGAL as a sensitive biomarker of AKI to screen and rank order compounds in a 2-day toxicity study. To our knowledge, this study provides a first example of successfully applying biomarkers of AKI in drug development.