Background: A microscopic analysis of tissue is the gold standard for cancer detection. Hematoxylin-eosin (HE) for the reporting of prostate biopsy (PB) is conventionally based on fixation, processing, acquisition of glass slides, and analysis with an analog microscope by a local pathologist. Digitalization and real-time remote access to images could enhance the reporting process, and form the basis of artificial intelligence and machine learning. Fluorescence confocal microscopy (FCM), a novel optical technology, enables immediate digital image acquisition in an almost HE-like resolution without requiring conventional processing. Objective: The aim of this study is to assess the diagnostic ability of FCM for prostate cancer (PCa) identification and grading from PB. Design, setting, and participants: This is a prospective, comparative study evaluating FCM and HE for prostate tissue interpretation. PBs were performed (March to June 2019) at a single coordinating unit on consecutive patients with clinical and laboratory indications for assessment. FCM digital images (n = 427) were acquired immediately from PBs (from 54 patients) and stored; corresponding glass slides (n = 427) undergoing the conventional HE processing were digitalized and stored as well. A panel of four international pathologists with diverse background participated in the study and was