Glycoproteins, such as Galectin-3 (Gal-3) and Prostate Specific Membrane Antigen (PSMA), are functional proteins involved in numerous biological activities that include cell apoptosis, angiogenesis, and inflammation. Downregulation of both in the highly metastatic human Prostate Cancer (CaP) cell line PC-3 reduces tumor growth. We used Raman Spectroscopy (RS) to examine the effect of a potent Gal-3 inhibitor (GB1107) in CaP cell lines of varying metastatic potential, namely PC-3, DU-145 and LNCaP. All three cancer lines had distinct Raman signatures. Raman spectra from PC-3, DU-145 and LNCaP cells treated with GB1107, compared to the untreated cells as controls, showed significant differences corresponding to changes in phosphatidylinositol (peak at 596 cm-1), O-P-O stretching DNA (786 cm-1), lipid/phospholipid DNA backbone (1090-1100 cm-1), nucleic acid, lipid, protein (amide III) (1296-1305 cm-1), fatty acid (1440 cm-1), and protein (amid I) (1655 cm-1), suggesting that DNA phosphate backbone may become unstable with cancer progression, facilitating cancer cell metastasis. Our data suggests that Gal-3 inhibitor induces significant alterations in major biochemical constituents, such as lipids, proteins, and nucleic acids, which may lead to structural and molecular changes in the cancerous prostate tissue. To further analyze these spectral differences, Singular Value Decomposition (SVD) and Linear Discriminant Analysis (LDA) were applied for classification, enabling effective differentiation between treated and untreated CaP cell lines. This highlights the therapeutic potential of Gal-3 inhibitor in prevention of CaP progression and metastases.