Gentamicin (GM) is an aminoglycoside antibiotic used in treatment of various types of bacterial infections, but the major adverse effect is drug-induced nephrotoxicity. This study aimed to determine biomarkers that might predict nephrotoxicity initiated by GM using serum or urinary proton nuclear magnetic resonance (1H NMR) spectral data in male Sprague-Dawley rats. GM (0, 30, or 300 mg/kg/d) was intraperitoneally administered for 3 consecutive days. Animals were sacrificed 2 d (D2) or 8 d (D8) after last administration of GM in order to perform analysis of serum biochemistries and histopathologic examination. Urine samples were collected every 24 h from prior to treatment until sacrifice. Serum and urinary 1H NMR spectral data revealed apparent differential clustering between control and GM-treated groups as evidenced by principal component analysis (PCA) and orthogonal projections to latent structure-discriminant analysis (OPLS-DA) in global and targeted profiling. The concentrations of endogenous serum metabolites including 3-hydroxybutyrate, alanine, citrate, creatine, glucose, and glycine were increased significantly on D2 or D8. Urinary levels of glucose, glycine, and succinate were significantly elevated on D2 or D8, whereas the concentration of hippurate was significantly decreased on D2 and D8. Correlation of serum and urinary 1H NMR OPLS-DA with serum biochemistry and renal histopathologic changes suggests that 1H NMR urinalysis may be used to reliably predict or screen for GM-induced nephrotoxicity. In contrast, Western blot analysis of kidney injury molecule-1 (KIM-1) demonstrated that protein expression was not markedly altered indicating this biomarker was not sensitive to detect GM-mediated renal damage. Data suggest that these altered metabolites might serve as specific and sensitive biomarkers for GM-mediated renal damage.