Background and Purpose Vancomycin is one of the most common antibiotics administered in the hospital setting, yet acute kidney injury is a major limiting factor. Common combinations of antibiotics with vancomycin have been reported to worsen and improve vancomycin-induced kidney injury. We aimed to study the impact of flucloxacillin and imipenem-cilastatin on kidney injury when combined with vancomycin in our translational rat model. Experimental Approach Male Sprague-Dawley rats received allometrically scaled (1) vancomycin (2) flucloxacillin, (3) vancomycin+flucloxacillin, (4) vancomycin+imipenem-cilastatin, or (5) saline for 4 days. Vancomycin was administered intravenously and flucloxacillin or imipenem-cilastatin were administered intraperitoneally. Kidney injury was evaluated via drug accumulation and urinary biomarkers including urinary output, kidney injury molecule-1 (KIM-1), clusterin, and osteopontin. Relationships between vancomycin accumulation in the kidney and urinary kidney injury biomarkers were explored. Key Results Urinary output increased every study day for vancomycin+flucloxacillin; whereas in the vancomycin group it was elevated after the first dose only. In the vancomycin+flucloxacillin group, urinary KIM-1/24h increased on all days compared to vancomycin. In the vancomycin+imipenem-cilastatin group, urinary KIM-1/24h was decreased on days 1 and 2 compared to vancomycin. Similar trends were observed for clusterin. More vancomycin accumulated in the kidney with vancomycin+flucloxacillin compared to vancomycin and vancomycin+imipenem-cilastatin. The accumulation of vancomycin in the kidney tissue correlated with increasing urinary KIM-1 (4-parameter Hill Slope, R2=0.7985). Conclusion and Implications Vancomycin+flucloxacillin caused more kidney injury compared to vancomycin alone and vancomycin+imipenem-cilastatin in a translational rat model as determined by multiple kidney injury biomarkers. The combination of vancomycin+imipenem-cilastatin was nephroprotective.