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Aim The traditional three-level H&E sectioning protocol for prostate biopsies was developed for ultrasound-guided systematic sampling and predates lesion-targeted biopsy approaches. Multiparametrical MRI (mpMRI) has improved detection of clinically significant prostate carcinoma but has also increased biopsy volume, workload, costs and digital pathology requirements, including whole-slide imaging and artificial intelligence (AI) integration.Methods and results A retrospective institutional cost-yield analysis was performed on prostate biopsy cases at Beth Israel Deaconess Medical Center (2015–2022), comparing systematic 12-core biopsies with combined systematic and mpMRI-targeted biopsies. Case volume, tissue blocks and H&E slides were analysed. Per-slide costs were calculated, and additional digital pathology and AI workflow costs were estimated using published data and vendor pricing. Case volume increased by 27% over 7 years, while combined systematic and mpMRI-targeted protocols increased tissue blocks and H&E slides by 39%. The cost per H&E slide was $7.18, with digital pathology and AI workflows adding an estimated $4.30–5.00 per slide. In systematic 12-core biopsies, third-level sectioning identified four additional low-volume carcinomas at a cost of $2283 per diagnosis. In combined biopsies, two additional carcinomas were detected exclusively on the third level, both Grade Group 1, with a cost of $27 657 per diagnosis.Conclusion Routine three-level H&E sectioning in combined systematic and mpMRI-targeted prostate biopsies demonstrated low incremental diagnostic yield and substantial additional processing cost. A two-level protocol with selective third-level sectioning may preserve detection of clinically significant carcinomas in this institutional cohort, while reducing workload and cost in the digital pathology era.