Histopathological detection and quantitation of glycogen in situ are important for the assessment of glycogen storage diseases and different types of cancer. The current standard method for defining the regionality of glycogen rely almost exclusively on Periodic Acid-Schiff (PAS) staining, a workflow that lacks specificity and sensitivity. Herein, we describe a new and much improved workflow to detect microenvironmental glycogen in situ using enzyme-assisted matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). This method provides superior sensitivity and can elucidate the molecular features of glycogen structure, with 50 μm spatial resolution for a next-generation histopathological assessment of glycogen.