The snake venom gland is the place for the synthesis, storage, and secretion of a complex mixture of proteins and peptides, i.e., the venom. The morphology of the gland has been revealed by classical histology and microscopic studies. However, knowledge about the gland's cellular secretory and functional processes is still incomplete and has so far been neglected by the omics disciplines. We used autofocusing atmospheric-pressure matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) to investigate endogenous biomolecular distributions in the venom glands of the saw-scaled viper, Echis carinatus sochureki, employing different sample preparation methods. Fresh-freezing and formalin-fixation were tested for the gland to obtain intact tissue sections. Subsequently, MSI was conducted with 12 μm pixel resolution for both types of preparations, and the lateral distributions of the metabolites were identified. Experiments revealed that lipids belonging to the classes of PC, SM, PE, PS, PA, and TG are present in the venom gland. PC (32:0) and SM (36:1) were found to be specifically located in the areas where cells are present. The snake venom metalloprotease inhibitor pEKW (m/z 444.2233) was identified in the venom by top-down LC-MS/MS and localized by MALDI-MSI in the gland across secretory epithelial cells. The peptide can inhibit the venom's enzymatic activity during long-term storage within the venom gland. With a high degree of spectral similarities, we concluded that formalin-fixed tissue, in addition to its high ability to preserve tissue morphology, can be considered as an alternative method to fresh-frozen tissue in the case of lipid and peptide MS imaging in venom gland tissues.