Glycosylation of cell surface, secreted, and circulating proteins is one of the most common types of post-translational modification. These modifications occur most commonly as one of three major classes: N-linked glycosylation on asparagine residues, O-linked glycosylation on serine or threonine residues, or as glycosaminoglycan oligosaccharide polymers on serine. Specifically, for N-linked glycans, an endoglycosidase enzyme, peptide N-glycosidase F (PNGase F), cleaves the attached oligosaccharides between the asparagine and first sugar. A method to analyze released N-glycans and map them to specific locations within a tissue is presented here. The PNGase F is applied by solvent sprayer as a molecular layer on frozen or formalin-fixed tissues and all released N-glycans in a given region of tissue are detected using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (MALDI-IMS). Using the described MALDI-IMS protocol, at least 40 or more individual N-glycans can be mapped to tissue histopathology and extracted for further structural analysis approaches. © 2018 by John Wiley & Sons, Inc.