Glycolysis is fundamental for cerebral energy metabolism. The current glycolysis pathway gives direct information about the local cellular health. To investigate hippocampal glycolysis in living rats, we established an experimental setup combining MRS and laser spectroscopy. It provides the opportunity to observe the behavior to the involved substances (glucose, lactate, NADH) for different glycolytic pathways. Under hyperglycemic conditions, blood oxygen limits glucose consumption leading to drops in NADH and lactate. In contrast, NADH and lactate concentrations increase under anaerobic conditions. This corresponds well with known literature and demonstrates the power of the presented setup to characterize hippocampal glycolysis in vivo.