IMPORTANCE: Historically, early detection of small asymptomatic kidney tumors presages better patient outcome. Screening for asymptomatic renal tumors by abdominal imaging is not cost-effective and cannot reliably distinguish benign from malignant tumors. OBJECTIVE: This investigation evaluated the clinical utility, sensitivity, and specificity of urine aquaporin-1 (AQP1) and perilipin-2 (PLIN2) concentrations as unique, noninvasive biomarkers to diagnose malignant clear cell or papillary renal cell carcinoma (RCC) in a screening paradigm. DESIGN, SETTING, AND PARTICIPANTS: From February through December 2012, urine samples were obtained from 720 patients undergoing routine abdominal computed tomography (CT) (screening population), 80 healthy controls, and 19 patients with pathologically confirmed RCC. MAIN OUTCOMES AND MEASURES: Urine AQP1 and PLIN2 concentrations were measured by sensitive and specific enzyme-linked immunosorbent assay and Western blot procedures, respectively, in all groups. In the otherwise asymptomatic screening population, the absence or presence of a renal mass and RCC were verified by abdominal CT and by postnephrectomy pathologic diagnosis, respectively. RESULTS: Urine AQP1 and PLIN2 concentrations were significantly higher (all P < .001) in the 19 patients with known RCC (AQP1 median [95% CI], 225.0 [121.0-450.0] ng/mg urine creatinine; and PLIN2 median [95% CI], 37.8 [22.8-83.7] absorbance units/mg creatinine) than in the 80 healthy controls (AQP1 median [95% CI], 1.1 [0.9-1.3] ng/mg urine creatinine; and PLIN2 median [95% CI], 3.1 [2.4-3.7] absorbance units/mg creatinine) and the 720 patient screening population (AQP1 median [95% CI], 0.5 [0.0-1.0] ng/mg urine creatinine; and PLIN2 median [95% CI], 0 [0-0] absorbance units/mg creatinine). The area under the receiver operating characteristic curve for urine AQP1 and PLIN2 concentrations individually or in combination was 0.990 or greater, with 95% or greater sensitivity and 91% or greater specificity compared with controls or the screening population. Of the 720 screened patients, 3 had biomarker concentrations suggestive of RCC and were found to have an imaged renal mass by CT. Two of the patients had pathologically confirmed RCC in further evaluation. CONCLUSIONS AND RELEVANCE: These results demonstrate the clinical utility, specificity, and sensitivity of urine AQP1 and PLIN2 to diagnose RCC. These tumor-specific proteins have high clinical validity and substantial potential as specific diagnostic and screening biomarkers for clear cell or papillary RCC and in the differential diagnosis of imaged renal masses.