Only 30% of patients with elevated serum prostate specific antigen (PSA) levels who undergo prostate biopsy are diagnosed with prostate cancer (PCa). Novel methods are needed to reduce the number of unnecessary biopsies. We report on the identification and validation of a panel of 12 novel biomarkers for prostate cancer (PCaP), using CE coupled MS. The biomarkers could be defined by comparing first void urine of 51 men with PCa and 35 with negative prostate biopsy. In contrast, midstream urine samples did not allow the identification of discriminatory molecules, suggesting that prostatic fluids may be the source of the defined biomarkers. Consequently, first void urine samples were tested for sufficient amounts of prostatic fluid, using a prostatic fluid indicative panel (“informative” polypeptide panel; IPP). A combination of IPP and PCaP to predict positive prostate biopsy was evaluated in a blinded prospective study. Two hundred thirteen of 264 samples matched the IPP criterion. PCa was detected with 89% sensitivity, 51% specificity. Including age and percent free PSA to the proteomic signatures resulted in 91% sensitivity, 69% specificity.