Mechanisms of pruritus are implicated in the dysregulation of the metabolites in the spinal cord. We investigated pruritus behavioral testing in three groups of young adult male C57Bl/6 mice, including one group treated with normal saline, while the other groups intradermally injected with alpha-Me-5-HT (histamine-independent pruritogen), compound 48/80 (histamine-dependent pruritogen) at the nape skin of the neck, respectively. Proton nuclear magnetic resonance spectroscopy (MRS) was used to compare spinal metabolites from the vertebral cervical among three groups, and to study the association of spinal metabolite ratio and pruritus intensity. The MRS-measured N-acetylaspartate-to-myoinositol ratio (NAA/Ins) was significantly correlated with the number of scratches between normal saline group and 48/80 group or alpha-Me-5-HT group (bothP<0.0001), indicating that NAA/Ins may be a robust surrogate marker of histamine-independent/dependent pruritogen. There was significant difference in Glu/Ins between normal saline group and 48/80 group (P=0.017), indicating that Glu/Ins may be a surrogate marker of histamine-dependent pruritogen, while GABA/Ins was highly significantly different between normal saline group and alpha-Me-5-HT group (P=0.008), suggesting that GABA/Ins may be a surrogate marker of histamine-independent pruritogen. MRS may reflect the extent of pruritus intensity elicited by alpha-Me-5-HT and compound 48/80 with sensitivity similar to the number of scratches, and above potential markers need to be further validated in pre-clinical and clinical treatment trials.