In vivo Magnetic Resonance Spectroscopy is a useful tool to characterize brain biochemistry as well as its alteration in a large number of major central nervous system diseases. The present review will focus on the study of the glutamate-glutamine cycle, an important biochemical pathway in excitatory neurotransmission, analyzed using in vivo MRS of different accessible nuclei: (1)H, (13)C, (15)N and (31)P. The different methodological aspects of data acquisition, processing and absolute quantification of the MRS data for each nucleus will be presented, as well as the description of the mathematical modeling approach to interpret the MRS measurements in terms of biochemical kinetics. The unique advantages of MRS, especially its non-invasive nature enabling longitudinal monitoring of brain disease progression and/or effect of treatment is illustrated in the particular context of hyperammonemic disorders with a specific focus on animal models. We review the current possibilities given by in vivo MRS to investigate some of the molecular mechanisms involved in hyperammonemic disorders and to give a better understanding of the process of development of hepatic encephalopathy, a severe neuropsychiatric disorder that frequently accompanies liver disease.