Label-free quantitative proteomics was employed to compare the protein content of extracellular vesicles isolated by various differential centrifugation-based approaches from expressed prostatic secretions in urine (EPS-urine) from men with prostate cancer. The developed optimized approach improved EV purity by depleting the high-abundance urine protein Tamm-Horsfall protein (THP) and other common contaminants and achieved relative enrichment of prostate cancer-associated EV-resident proteins.