Earlier and more reliable detection of drug-induced kidney injury would improve clinical care and help to streamline drug-development. As the current standards to monitor renal function, such as blood urea nitrogen (BUN) or serum creatinine (SCr), are late indicators of kidney injury, we conducted ten nonclinical studies to rigorously assess the potential of four previously described nephrotoxicity markers to detect drug-induced kidney and liver injury. Whereas urinary clusterin outperformed BUN and SCr for detecting proximal tubular injury, urinary total protein, cystatin C and beta2-microglobulin showed a better diagnostic performance than BUN and SCr for detecting glomerular injury. Gene and protein expression analysis, in-situ hybridization and immunohistochemistry provide mechanistic evidence to support the use of these four markers for detecting kidney injury to guide regulatory decision making in drug development. The recognition of the qualification of these biomarkers by the EMEA and FDA will significantly enhance renal safety monitoring.