The prostate gland secretes many proteins in a prostatic fluid that combines with seminal vesicle derived fluids to promote sperm activation and function. Proximal fluids of the prostate that can be collected clinically are seminal plasma and expressed-prostatic secretion (EPS) fluids. EPS represents the fluid being secreted by the prostate following a digital rectal prostate massage, which in turn can be collected in voided urine post-exam. This collection is not disruptive to a standard urological exam, and it can be repeatedly collected from men across all prostatic disease states. A direct EPS fluid can also be collected under anesthesia prior to prostatectomy. While multiple genetic assays for prostate cancer detection are being developed for the shed epithelial cell fraction of EPS urines, the remaining fluid that contains many prostate-derived proteins has been minimally characterized. Approaches to optimization and standardization of EPS collection consistent with current urological exam and surgical practices are described, and initial proteomic and glycomic evaluations of the of EPS fluid are summarized for prostate specific antigen and prostatic acid phosphatase. Continued characterization of the prostate specific protein components of EPS urine combined with optimization of clinical collection procedures should facilitate discovery of new biomarkers for prostate cancer.