The mechanism behind itching is not well understood. Proton nuclear magnetic resonance (H-1-NMR) spectroscopic analysis of spinal cord extracts provides a quick modality for evaluating the specific metabolic activity of alpha-Me-5-HT-evoked pruritus mice. In the current study, four groups of young adult male C5781/6 mice were investigated; one group treated with saline, while the other groups intradermally injected with alpha-Me-5-HT (histamine independent pruritogen), histamine (histamine dependent pruritogen) and capsaicin (algogenic substance), respectively. The intradermal microinjection of alpha-Me-5-HT and histamine resulted in a dramatic increase in the itch behavior. Furthermore, the results of NMR studies of the spinal cord extracts revealed that the metabolites show very different patterns for these different drugs, especially when comparing alpha-Me-5-HT and capsaicin. All the animals in the groups of alpha-Me-5-HT and capsaicin were completely separated using the metabolite parameters and principal component analysis. For alpha a-Me-5-HT, the concentrations of glutamate, GABA, glycine and aspartate increased significantly, especially for GABA (increased 17.2%, p = 0.008). Furthermore, the concentration of NAA increased, but there was no significant difference (increased 11.3%, p = 0.191) compared to capsaicin (decreased 29.1%, p = 0.002). Thus the application of magnetic resonance spectroscopy technique, coupled with statistical analysis, could further explain the mechanism behind itching evoked by alpha-Me-5-HT or other drugs. It can thus improve our understanding of itch pathophysiology and pharmacological therapies which may contribute to itch relief.