Raman spectroscopy has been proposed for detecting biochemical alterations in prostate cancer (PrCa) compared to benign prostate tissue in in vitro fragments from surgery for diagnostic purposes. Freezer-stored fragments of human prostate tissues were unfrozen and submitted to Raman spectroscopy with a dispersive spectrometer (830-nm and 200-mW laser parameters, 30-s exposure time). Fragments were fixed and submitted to histopathology to grade PrCa according to Gleason score. A total of 160 spectra were taken from 32 samples (16 benign tissues and 16 PrCa tissues). The relative concentrations of selected biochemicals were estimated using a least-squares fitting model applied to the spectra of pure compounds and the tissue spectrum. A discrimination model was developed employing the most statistically relevant compounds with capability of separating PrCa from benign tissues. The fitting model revealed that actin, hemoglobin, elastin, phosphatidylcholine, and water are the most important biochemicals to discriminate prostate depending on the Gleason score. A discrimination based on Euclidean distance using the relative concentrations of phosphatidylcholine and water showed the higher accuracy of 74 % to discriminate PrCa from benign tissue. Raman spectroscopy is an analytical technique with possibility for identifying biochemical constitution of prostate and could be used for diagnostic purposes.