SUMMARY Rapid, reliable diagnosis of prostate cancer (PCa) is highly desirable as current methods for diagnosis lack sensitivity to successfully detect this type of cancer in early stages. In addition, identification of PCa biomarkers that can classify patients into high- and low-risk groups for disease progression may impact treatment decision-making and is a major area of on-going research. Here, we describe a set of protein-combination panels in urinary extracellular vesicles (EVs), defined by targeted proteomics and immunoblotting techniques, to improve early non-invasive detection and stratification of PCa patients. We report a two-protein combination in urinary EVs to classify benign and PCa patients (ADSV-TGM4; AUC= 0.652), and a combination of five proteins able to significantly distinguish between high- and low-grade PCa patients (CD63-GLPK5-SPHM-KLK3-PAPP; AUC=0.701). Immunohistochemistry assays on 98 benign prostatic versus 136 PCa tissues confirmed a strong link between the urinary EVs proteome and expression alterations in PCa tissues. ADSV and TGM4 expression yielded a high diagnostic potential (AUC 0.729 and 0.813) and promising TGM4 prognostic power between low and high-grade tumors (AUC 0.817) as well as detecting those patients with biochemical recurrence (AUC = 0.803). Our study highlights the potential of SRM as diagnostic assay for liquid biopsies via urinary EVs to improve diagnosis and prognosis of suspected PCa patients.