Vancomycin, a first-line drug for treating methicillin-resistant Staphylococcus aureus infections, is associated with acute kidney injury (AKI). This study involved an evaluation of biomarkers for AKI detection and their comparison with traditional serum creatinine (SCr). We prospectively enrolled patients scheduled to receive intravenous vancomycin for methicillin-resistant S aureus infection. Blood samples for pharmacokinetic assessment and SCr and cystatin C (CysC) measurements were collected at baseline and on days 3, 7, and 10 from the initiation of vancomycin administration (day 1). Urinary biomarkers, including kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin, and clusterin, were collected from days 1 to 7 and adjusted for urinary creatinine levels. The estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation. Of the 42 patients, 6 experienced vancomycin-induced AKI. On day 7, the change from baseline eGFR using CysC (ΔeGFR_CysC) showed a stronger correlation with vancomycin area under the curve (r = −0.634, P < .001) than that using SCr (ΔeGFR_SCr; r = −0.437, P = .020). ΔeGFR_SCr showed no significant correlation with vancomycin pharmacokinetic in patients with body mass index ≥23. The median (interquartile range) level of KIM-1 (μg/mg) was significantly higher in the AKI group (0.006 [0.005–0.008]) than in the non-AKI group (0.004 [0.001–0.005]) (P = .039, Mann–Whitney U test), with area under the receiver operating characteristic curve (95% confidence interval) of 0.788 (0.587–0.990). Serum CysC, particularly in overweight individuals or those with obesity, along with urinary KIM-1 are important predictors of vancomycin-induced AKI. These results may aid in selecting better biomarkers than traditional SCr for detecting vancomycin-induced AKI.