INTRODUCTION AND OBJECTIVES: Prostate cancer is currently screened by an imperfect serum PSA test. Since PSA is not specific to cancer, this leads to overdiagnosis where men undergo unnecessary biopsies to find no cancer, and overtreatment for tumors that may not be life threatening. Better markers are needed. Using cell type-specific transcriptomes, genes with increased expression in cancer cells (of Gleason patterns 3 and 4) and cancer-associated stromal cells (of different Gleason scores) were identified, and those encoding secreted proteins were selected for marker development: AGR2, AGR3, CCL3, CEACAM5, CEACAM6, CRISP3, CXCL14, IL24, MMP9, POSTN, SFRP4, CD90. METHODS: Voided urine samples were collected from consented patient donors at pre-op, and healthy controls with no diagnosis of cancer. The urine was spin concentrated to 1 ml and the protein concentration was estimated by BCA Protein Assay. PRISM-SRM (high pressure high-resolution separations coupled with intelligent selection and multiplexing-selective reaction monitoring) targeted proteomics was used to measure simultaneously these protein biomarkers in individual samples. The marker amounts were presented as ratios of PSA, which was used for 0normalization0. In data analysis, cancer severity ranking was generated from pathology parameters of the tumors including Gleason score, tumor volume and stage. Statistical analysis and graphical display of the data were carried out with OriginPro 2015. RESULTS: The data from a cohort of 14 cases (UW) and 25 controls (UTSA + UW) was analyzed to produce AUC (area under the curve) values ranging from 0.774 for AGR2, 0.820 for CXCL14, 0.820 for CEACAM5, 0.837 for CD90, 0.871 for MMP9, 0.889 for SFRP4, 0.889 for IL24, 0.894 for CRISP3, to 0.906 for CEACAM6, and 0.914 for the marker panel of nine proteins (P1⁄42.20E-05) in distinguishing cancer from non-cancer. For correlation with tumor pathology, the CEACAM5/ CRISP3 concentration ratios showed increase with severity ranking (R1⁄40.5219) while the CD90/CRISP3 ratios showed decrease with ranking (R1⁄40.445). High CEACAM5/CRISP3 and low CD90/CRISP3 ratios were indicative of high risk disease. Other ratios had lower R values. CONCLUSIONS: For prostate cancer detection, voided urine provides the least invasive means for cancer detection. The AUC value obtained from the marker panel is superior to that of PHI (0.708) and MiPS (Michigan Prostate Score, 0.762). Urine collection is simple (at patients0 convenience) and does not require a vigorous DRE, which makes the test suitable for population screening. The marker ratios could also provide information on high risk vs. low risk regarding the cancer detected. A useful multimarker test could eventually be applied to replace biopsy.