To determine the optimal number of cores needed during microultrasound-informed prostate biopsy for the detection of clinically significant prostate cancer (csPCa, defined as Gleason Grade Group ≥2).A retrospective review of 1011 consecutive patients between September 2021 and July 2023 at our institution were identified; 536 underwent microultrasound biopsy and 475 underwent magnetic resonance imaging (MRI)/ultrasound (US) targeted biopsy. Lesions were given a Prostate Risk Identification using Microultrasound (PRI-MUS) score, with lesions PRI-MUS ≥3 targeted. MRI lesions were scored with Prostate Imaging-Reporting and Data System (PI-RADS) and lesions PI-RADS ≥3 were targeted. The primary outcome is the detection of csPCa stratified by number of cores.One hundred thirty-eight patients underwent targeted biopsies for microultrasound only lesions, 182 for microultrasound and MRI lesions and 426 underwent MRI/US for MRI lesions. The first targeted core detected 78.0% (46/59), 77.8% (63/81), and 78.8% (216/274) of csPCa for microultrasound, microultrasound+MRI, and MRI/US, respectively. Comparing first to third core, there was not a significant difference in overall detection of csPCa by microultrasound, though MRI/US was significantly different (28.4% vs 36.4% P = .12, 32.5% vs 41.8% P = .06, 42.5% vs 53.9% P < .001 for microultrasound, microultrasound+MRI, and MRI/US, respectively). PI-RADS 3 and PRI-MUS 3 lesions had lower first core detection rates compared to PI-RADS 5 and PRI-MUS 5 lesions (44.4% vs 85.4% P = .01, 65.2% vs 81.4% P = .14, 60% vs 83.1% P = .07 for microultrasound, microultrasound+MRI, and MRI/US, respectively).A three-core targeted biopsy per microultrasound lesion improves detection rate of csPCa and should be considered to improve diagnostic accuracy.