The liver serves as a hub for amino acid metabolism, in particular concerning ammonia detoxification. The mitochondrial enzyme glutamate dehydrogenase (GDH) plays a central role this pathway, bridging amino acid deamination and glutamate handling toward urea formation. Numerous studies have investigated the function of GDH in the liver, pointing to complex allosteric regulations. However, typical measurements of GDH expression and activity in tissue extracts do not allow the assessment of the actual in situ enzymatic activity that relies on the preservation of intracellular organization. In this study, we developed an in situ redox-sensitive nitro blue tetrazolium (NBT) assay to investigate the hepatic GDH activity in cryopreserved liver sections. On the same section series, immunohistochemistry provided corresponding mapping of the expression of GDH and associated enzymes. The study was conducted in control floxed mice as well as in liver-specific GDH knockout mice (Hep-Glud1-/-). We evaluated the responses to different dietary protein intakes, i.e. mice fed a standard 20% protein diet versus 30% and 45% protein contents as high protein diets. Tissues of interest were collected from control floxed mice and Hep-Glud1-/- mice after 4 hours of fasting. Measured in liver lysates of control floxed mice, expression and activity of GDH were not significantly changed by the high protein diets. However, measured in situ, the NBT-based assay revealed that GDH activity was markedly increased by the high protein diets. In vivo, this was accompanied by an increase in plasma urea levels, while plasma ammonia levels remained unchanged. Surprisingly, Hep-Glud1-/- mice lacking liver GDH exhibited hyperammonemia, even under the standard 20% protein diet. In control floxed mice, immunohistochemistry on liver cryosections showed that GDH was evenly distributed within the tissue. However, the NBT assay performed on the same cryosections revealed hot spots of in situ GDH activity predominantly in hepatic perivenous area. These readouts were absent in Hep-Glud1-/- mice. Overall, our study shows that commonly used assessments of GDH function do not reflect in situ activity. Such activity is increased by a high protein diet, while expression levels are unchanged; measurements of GDH activity in liver extracts missing the in situ adaptation of the enzyme uncovered by the NBT assay.