Prostate cancer is the most prevalent cancer in men over the age of 50 years. Prostate-specific antigen is limited as both an early detection and prognostic biomarker. Prostasomes are unique microvesicles of endocytic origin with a unique lamellar membrane composed of cholesterol and phospholipids known to be capable of fusing with other cells and thus acting as messengers between cells. The evaluated article presents a new highly sensitive and specific protein-targeted assay based upon a proximity ligation assay (PLA) deemed capable of detecting prostasomes in the blood plasma of men with prostate cancer. The 4-PLA assay was used to detect circulating prostasomes in men with prostate cancer compared with age-matched controls. The median prostatsome levels in blood plasma were 2.5- to seven-fold higher compared with controls. The blood plasma prostasome levels correlated with Gleason 7 and higher disease versus Gleason 6 or lower and controls. This study describes for the first time a highly sensitive assay that depends upon simultaneous binding to as many as five different epitopes for detection and is thus a new and powerful tool for biomarker identification and validation.