%p2PSA and prostate health index (phi) has shown valuable results in the detection of prostate cancer (PCa), improving the prediction of the aggressiveness of the tumor. The goal of the present study was to evaluate %p2PSA and phi in the detection of PCa, estimating their relationship with the aggressiveness of PCa. A total of 354 patients with positive or negative prostatic biopsy were included. Prospectively, 150 were enrolled and 204 were enrolled retrospectively proceeding from our serum bank. The best performance was observed for %p2PSA and phi, obtaining an AUC of 0.723 and 0.732, respectively. The highest specificity at sensitivity around 90% was obtained for phi (27.4%). Using the cut-off of 31.94 for phi, a reduction of 19% biopsies could be obtained, while 17 PCa would have been missed, including only four patients with a Gleason score ≥7. Similarly, using a cut-off of 1.21 for %p2PSA, a reduction of 12.7% biopsies could be obtained, while 16 PCa would have been missed, including only four patients with a Gleason score ≥7. Moreover, among patients with PCa, phi (median: 69.75 vs. 48.04) and %p2PSA (median: 2.60 vs. 1.98) values are significantly higher (p<0.0001) in patients with a biopsy Gleason score ≥7. Our results confirm previous evaluations, showing similar AUCs and results in sensitivity and specificity to other studies.%p2PSA and phi raise the accuracy in the detection of prostate cancer, reducing the number of unnecessary biopsies and improving the prediction of the aggressiveness of the tumor.