The Prostate CAncer gene 3 (PCA3) assay is a novel tool that might aid in the diagnosis of prostate cancer and that might indicate the significance of the disease. In this review we discuss five clinical cases in which the PCA3 assay can be considered, based on scientific evidence and key patient cases from real-life clinical practice. The PCA3 assay might be used to guide biopsy decisions in: (i) Men with an elevated serum total prostate specific antigen (tPSA) level and one or more previous negative biopsies; (ii) men with a normal tPSA level and a family history of prostate cancer; (iii) men with an elevated tPSA level (2.5-10 ng/mL) and no previous biopsy; (iv) men with an elevated tPSA level and a concomitant urinary condition. In addition, in men diagnosed with prostate cancer, the PCA3 assay could aid in the decision of whether active therapy is needed or active surveillance is appropriate.