PURPOSE: To assess the value of secondary lesion-targeted biopsy (SLx) in detecting prostate cancer (PCa) among patients with multifocal disease. METHODS: A total of 298 biopsy-naïve patients with 612 lesions (all with Prostate Imaging Reporting and Data System [PI-RADS] v2.1 ≥ 3) underwent cognitive fusion-targeted biopsy (TB) combined with systematic biopsy (SB). Our primary endpoints were to compare the detection rates of PCa and clinically significant PCa (csPCa) across different biopsy strategies (Index lesion-targeted biopsy [ILx] vs. ILx + SLx and ILx + SB vs. ILx + SLx + SB) and to define potential indications for SLx using PI-RADS and PSA density (PSAD). Secondary endpoint was to evaluate the predictive performance of index lesion (IL)- and SL-based multivariate logistic regression (MVA) models for csPCa. RESULTS: The overall detection rates for PCa and csPCa were 71% and 60%, with ILx + SLx + SB as the gold standard. Adding SLx to ILx modestly increased detection rates for PCa (63% vs. 65%, P = 0.016) and csPCa (55% vs. 58%, P = 0.004), but offered no significant advantage over ILx + SB. Stratification by PI-RADS and PSAD revealed that focusing on 80% intermediate- to high-risk lesions detected 39% csPCa while reducing 20% low-risk SLx at the cost of missing 1.6% csPCa. IL-based models outperformed SL-based models in predicting csPCa (Hosmer-Lemeshow P = 0.653 vs. 0.461). CONCLUSION: SLx provides limited benefit in csPCa detection when ILx and SB have already been performed. Combining PI-RADS scores and PSAD helps identify patients who could benefit from SLx while avoiding unnecessary procedures in low-risk cases. CLINICAL TRIAL REGISTRATION: No. 2016 - 1252, January 2017.