Mass spectrometry–based investigation of the heterogeneous glycoproteome from complex biological specimens is a robust approach to mapping the structure, function, and dynamics of the glycome and proteome. Sampling whole wet tissues often provides a large amount of starting material; however, there is a reasonable variability in tissue handling prior to downstream processing steps, and it is difficult to capture all the different biomolecules from a specific region. The on-slide tissue digestion approach, outlined in this protocol chapter, is a simple and cost-effective method that allows comprehensive mapping of the glycoproteome from a single spot of tissue of 1 mm or greater diameter. It provides a selection of target areas on tissue slides appropriate for tissue volumes of 10 nL or greater, corresponding to a 1 μL droplet of enzyme solution applied to a 1-mm diameter target on a 10-μm-thick tissue slice. Sequential enzymatic digestions and desalting of the biomolecules without any prior derivatization from the surface of fresh frozen or formalin-fixed paraffin-embedded tissue slides enable the simultaneous identification of glycosaminoglycan disaccharides such as hyaluronan, chondroitin sulfate and heparan sulfate, asparagine or N-linked glycans, and intact (glyco)peptides using liquid chromatography-tandem mass spectrometry. The in-depth information obtained from this method including the disaccharide compositions, glycan structures, peptide abundances, and site-specific glycan occupancies provides a detailed profiling of a single spot of tissue which has the potential to be disseminated to biomedical laboratories.