In this study, we aimed to validate the Prostate Health Index (PHI) for the detection of prostate cancer (PCa). We prospectively enrolled patients aged 50-75 years with a serum prostate specific antigen (PSA) level of 4-10 ng/mL undergoing transrectal biopsy of the prostate between April 2016 and May 2017. The primary outcome was the diagnostic performance of various PSA derivatives (total PSA, free PSA, %fPSA, p2PSA, %p2PSA, and PHI) to predict PCa. The secondary outcome was comparisons of PSA derivatives between patients with a Gleason score (GS) ≤6 and GS ≥7. PCa was diagnosed in 36 of 154 (23.4%) patients, and 26 (16.9%) had a GS ≥7. The areas under the receiver operating characteristic curves were significantly greater in %p 2PSA and PHI than in PSA (0.76 vs. 0.57, p = 0.015 and 0.77 vs. 0.57, p = 0.004, respectively). Patients with a GS ≥7PCa had marginally higher %p2PSA and PHI than those with a GS of 6 (17.8 vs. 12.73, p = 0.06; 46.58 vs. 31.55, p = 0.05). At a PHI cutoff value of 29.6, the sensitivity and specificity were 77.8% and 67.8% in detecting PCa, respectively. In addition, 57.1% of the patients avoided an unnecessary biopsy, while three patients (1.9%) with GS 7 PCa were missed. In conclusion, the ability of %p2PSA and PHI to predict prostate biopsy outcome was better than that of PSA and %fPSA in the initial biopsy in Taiwanese men with serum PSA between 4 and 10 ng/mL.