In this clinical validation study, we developed and validated a urinary Q-Score generated from the quantitative test QSant, formerly known as QiSant, for the detection of biopsy-confirmed acute rejection in kidney transplants. Using a cohort of 223 distinct urine samples collected from three independent sites and from both adult and pediatric renal transplant patients, we examined the diagnostic utility of the urinary Q-Score for detection of acute rejection in renal allografts. Statistical models based upon the measurements of the six QSant biomarkers (cell-free DNA, methylated-cell-free DNA, clusterin, CXCL10, creatinine, and total protein) generated a renal transplant Q-Score that reliably differentiated stable allografts from acute rejections in both adult and pediatric renal transplant patients. The composite Q-Score was able to detect both T cell-mediated rejection and antibody-mediated rejection patients and differentiate them from stable non-rejecting patients with a receiver-operator characteristic curve area under the curve of 99.8% and an accuracy of 98.2%. Q-Scores < 32 indicated the absence of active rejection and Q-Scores ≥ 32 indicated an increased risk of active rejection. At the Q-Score cutoff of 32, the overall sensitivity was 95.8% and specificity was 99.3%. At a prevalence of 25%, positive and negative predictive values for active rejection were 98.0% and 98.6%, respectively. The Q-Score also detected subclinical rejection in patients without an elevated serum creatinine level but identified by a protocol biopsy. This study confirms that QSant is an accurate and quantitative measurement suitable for routine monitoring of renal allograft status.