Background: PSA and DRE are used for biopsy decisions; however, limited sensitivity and specificity have resulted in the over-diagnosis and treatment of low risk prostate cancer (PCa). Non-invasive screening tools that add predictive value for identifying high-grade, Gleason score (GS) ≥ 7, should impact the current paradigm. We sought to evaluate performance of a gene signature that differentiated GS7+ from GS6 + benign disease by evaluating patients with first-time biopsy results and gray zone PSA (4-10ng/mL) from an ongoing prospective clinical trial. Methods: 499 sequentially obtained first-catch non-DRE urine specimens (standard cups) from 15 urology practices were collected from patients presenting for biopsy. Exosomes were isolated and RNA extraction performed. RT-qPCR RNA copy numbers of ERG and PCA3, normalized to SPDEF, were measured to generate a three-gene signature, defined to yield a score discriminating GS7+ vs. GS6 and benign lesions, evaluating NPV, Sensitivity and Specificity relative to standard of care. Results: Urine samples from 205 men of 499 total (PSA 4-10 ng/mL, first biopsy, < 50 mL donation volume; median age 64 years, median PSA 5.46 ng/mL, 80% negative DRE, 74% no family history, 68% Caucasian), were evaluated. With 50% and 33% prevalence of positive biopsy for any cancer or GS7+ disease, respectively, a dichotomous gene signature + standard of care demonstrated good clinical performance in predicting biopsy result. For GS7+, with a 90% fixed sensitivity, the NPV and PPV were 92% and 48%, respectively compared to 79% and 35% for the prostate cancer prevention trial risk calculator (PCPTRC). A continuous score alone had an AUC of 0.74 for discriminating GS7+ from GS6 and benign disease and the results were significantly better than PCPTRC, AUC 0.64(p = 0.026). Conclusions: We confirmed a novel, non-invasive urine exosome gene signature demonstrated independent, negative predictive value for the diagnosis of GS7+ from first biopsy patients with ‘gray zone’ PSA. Its use in the biopsy decision process should result in fewer prostate biopsies pending completion of the prospective trial.