Renal cell carcinoma (RCC) remains a difficult-to-detect cancer for which new detection methodologies are urgently needed to improve screening efficacy. Urinary metabolomic profiling has identified several potential RCC biomarkers, including quinolinic acid, 4-hydroxybenzoic acid, gentisic acid and aketoglutaric acid. These four endogenous metabolites have unique pathophysiological mechanisms associated with RCC, suggesting that they may be promising biomarkers for earlier RCC detection.However, the lack of targeted analytical methods for their simultaneous determination in urine has hindered efforts to evaluate their clinical applicability. In this study, a robust high-performance liquid chromatography -tandem mass spectrometry (HPLC-MS/MS) technique using multiple reaction monitoring was developed to simultaneously monitor these four urinary biomarkers. The method was validated by multiple figures of merit including method detection limits (0.05 ng mL À1 to 1 ng mL À1 ), spiked recovery accuracy (88.6-114.8%) and reproducibility (0.4-9.3% RSD). The applicability of the method was further demonstrated in clinically relevant 100-fold diluted urine specimens. All four potential biomarkers were successfully quantified with urinary concentrations that were in agreement with previous reports. In conclusion, an accurate and sensitive method has been described for the simultaneous determination of quinolinic acid, 4-hydroxybenzoic acid, gentisic acid and a-ketoglutaric acid. We anticipate this method to significantly benefit clinical translational research aiming to evaluate the clinical applicability of these four potential biomarkers.