Metabolomics enables the provision of sensitive bio-markers of disease. We performed 800 MHz ¹H-nuclear magnetic resonance (NMR) spectroscopic analyses of cerebrospinal fluid (CSF) specimens to identify biomarkers of multiple sclerosis (MS), yielding reproducible detection of 15 metabolites from MS (n=15) and non-MS (n=17) patients. Mean levels of choline, myo-inositol and threonate were increased, whereas 3-hydroxybutyrate, citrate, phenylalanine, 2-hydroxyisovalerate and mannose were decreased in MS-derived CSF (p<0.05), suggesting alterations to energy and phospholipid metabolism. Multivariate hierarchal cluster analysis indicated a high correlation within the metabolite profiles, significantly clustering samples into the two clinical groups, which was corroborated using principal components analysis. CSF metabolomics have the capacity to yield quantitative biomarkers and insights into the pathogenesis of MS.