INTRODUCTION: Maximizing surgical resection in gliomas is associated with a survival benefit, which can be negated by surgery-inflicted neurological deficits, making it imperative to achieve extensive resection without compromising non-infiltrated tissue. Current technologies are sub-optimal in providing intra-operative molecular characterization. METHODS: This is a prospective cohort study using intraoperative DESI-MS analysis of smears produced from freshly obtained brain tumor biopsy samples under neuro-navigation guidance to evaluate IDH mutation status via 2-hydroxyglutarate (2-HG) intensity and TCP via measurement of N-acetylaspartic acid (NAA) intensity and characteristic lipid profiles. Blinded review of the smears by a senior neuropathologist was used for validation of IDH mutation status and TCP estimates. RESULTS: A total of 465 tumor biopsies from 77 enrolled patients have been collected and analyzed. Evaluation of TCP in an optimization stage based on NAA intensity in 203 biopsies yielded sensitivity, specificity, and accuracy values of 91, 76, and 83%, while assessment through lipid profiling yielded 76, 85, and 81%, respectively. Assessment of IDH mutation status from 71 core biopsies resulted in sensitivity, specificity, and accuracy values of 89, 100 and 94%, respectively. Further optimization of IDH assessment by measurement of the relative intensity of 2-HG against its endogenous standard, glutamate, in nine patients yielded 100% sensitivity, specificity, and accuracy. CONCLUSION: We present preliminary results from the first and largest study using DESI-MS to intraoperatively evaluate TCP and IDH mutation status in gliomas. Prospectively, we propose a further modification to allow estimation of TCP and IDH mutation status in surgical cavities without the need of a biopsy by placing a surgical material (e.g. cottonoid) along the surgical margin and transferring material from the blot to a microscope slide prior to DESI-MS analysis.