Prostate cancer is one of the most common malignant tumors and a leading cause of cancer-related morbidity and mortality. Irrespective of the method that allows for risk stratification of prostate cancer suspects, diagnosis relies on tissue sampling through prostate biopsy and subsequent histopathological evaluation. This provides critical information about disease aggressiveness, which is required for adequate patient management. Prostate biopsy methods have significantly evolved over the years, including the definition of indications, sampling schemes and use of imaging techniques (ultrasound and MRI) that allow for more accurate tissue sampling. In response to the challenges emerging from more precise collection of minute prostate tissue samples for analysis, histopathological assessment should include not only the observation of routinely stained sections, but also, and increasingly so, a series of ancillary techniques, especially immunohistochemistry, which increment the accuracy of prostate cancer diagnosis and may provide relevant information to guide patient management.