Glutamate dehydrogenase (GDH) is a mitochondrial enzyme that regulates central metabolic pathways connecting the key metabolites. GDH activity is tissue-specific and allosterically regulated. The usual assessment of GDH activity in various organs requires extraction and homogenization of the tissue, thereby losing its in situ organization and specific distribution. This study developed in situ redox-sensitive nitro blue tetrazolium (NBT) enzymatic assay coupled with Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) metabolomics on cryopreserved tissue sections to investigate GDH regulation in the liver and pancreatic islets under different metabolic conditions. In pancreatic beta-cells, we revealed cataplerotic glutamate production by GDH during stimulation of insulin secretion. In the liver, we uncovered the pivotal role of hepatic GDH in modulating nitrogen and glucose homeostasis, as well as a circadian response to dietary protein source changes. Our study underscores the central role of GDH in amino acid metabolism in the liver and pancreatic beta-cells.