Malignant transformation of the extracellular matrix (ECM) disrupts the homeostatic tissue microenvironment, altering tissue processes of adhesion, cell death, migration, and proliferation to promote tumor growth. The cellular glycocalyx, comprised of multiple types of glycans represented by N-linked and O-linked glycoproteins, glycosphingolipids, and glycosaminoglycans, in turn interacts with multiple ECM proteins in the stroma, as well as immune system components. We hypothesize that characterization of co-localized glycosylation and ECM protein changes in the prostate tumor microenvironment could lead to novel diagnostic and therapeutic biomarkers of PCa across the clinical spectrum of the disease. A combined MALDI imaging mass spectrometry analysis workflow has been used to characterize FFPE prostate tissue slices representative of the clinical spectrum of PCa progression. Tissues are processed initially for release of N-glycans by spraying a molecular coating of peptide N-glycosidase F (PNGase F) enzyme via solvent sprayer, followed by analysis on a 7T MALDI-FTICR mass spectrometer. Analytes and matrix are removed, followed by digestion of the tissue with sprayed bacterial collagenase, and additional analysis by MALDI-FTICR MS. Tissue images are processed using FlexImaging and SCiLS Lab software. Detected glycan masses are cross-referenced with an in-house N-glycan structural database. Total numbers of N-glycans detected for PCa tissues are generally n = 60-70. Additional glycan analysis of adjacent tissue slides is feasible to assess sialic acid N-glycan isomers using chemical amidation, and fucosylated N-glycan isomers using a core fucose specific endoglycosidase, Endo F3. Collagenase-derived ECM peptides are compared to an in-house database of prostate ECM peptides, determined separately by LC-MS, which currently represents 87 individual PCa tissue proteins (68% ECM proteins with 18 collagen sub-types, 10% membrane proteins). Multiple MS images can be overlayed with H&E stained tissue to histopathology co-localize N-glycans and ECM proteins associated with tumor, stroma, hyperplasia and normal gland regions.