The purpose of this study was to confirm the exactitude of in vitro nuclear magnetic resonance spectroscopy(NMRS) and to complement the defect of in vivo NMRS. It has been difficult to understand the metabolism of a cerebellum using in vivo NMRS owing to the generated inhomogeneity of magnetic fields (B0 and B1 field) by the complexity of the cerebellum structure. Thus, this study tried to more exactly analyze the metabolism of a canine cerebellum using the cell extraction and high resolution NMRS. In order to conduct the absolute metabolic quantification in a canine cerebellum, the spectrum of our phantom included in various brain metabolites (i.e., NAA, Cr, Cho, Ins, Lac, GABA, Glu, Gln, Tau and Ala) was obtained. The canine cerebellum tissue was extracted using the methanol-chloroform water extraction (M/C extraction) and one group was filtered and the other group was not under extract processing. Finally, NMRS of a phantom solution and two extract solution (90% D2O) was progressed using a 500MHz (11.4 T) NMR machine. Filtering a solution of the tissue extract increased the signal to noise ratio (SNR). The metabolic concentrations of a canine cerebellum were more close to rat’s metabolic concentration than human’s metabolic concentration. The present study demonstrates the absolute quantification technique in vitro high resolution NMRS with tissue extraction as the method to accurately measure metabolite concentration.