Objectives We studied the frequency, inter-pathologist variation, appropriateness and utility of immunohistochemistry (IHC) performed on prostate biopsies (PB) to determine the significance of foci of suspicious glands/atypical small acinar proliferations (ASAP). Methods We calculated the rate of IHC use and diagnostic rate of ASAP and adenocarcinoma in PB from 01/01/2008 to 06/30/2015 for individual pathologists working in a tertiary academic institution, and correlated them with the pathologists’ experience, subspecialization and PB volume with the aim of determining the interpathologist variation and appropriateness of use of IHC according to recently published recommendations, and the usefulness of IHC to resolve foci of ASAP as either benign or adenocarcinoma. Results IHC was used in 966/2652 (36.4%, 95% CI 33.4–39.4%) PB cases and 1915 of 16,359 (11.7%, 95% CI 11.2%–12.2%) of PB blocks and allowed definitive diagnosis of either benign or malignant in 75.8% (95% CI 73.9–77.7%) of blocks. By pathologist, IHC use rates varied more than twofold (22.8–50.5%); higher use was found for pathologists with genitourinary pathology specialization, higher PB volume and more experience, and correlated with higher rates of both ASAP and adenocarcinoma diagnoses. The use of IHC stains was considered appropriate in 822/966 (85.1%, 95% CI 82.9–87.4%) cases. Conclusions Despite the fact that the use of IHC stains was considered useful and deemed appropriate in the majority of cases, it showed wide variation between pathologists, suggesting monitoring of IHC use rates may be useful to standardize its use.