Despite significant advances in image-guided therapy, surgeons are still too often left with uncertainty when deciding to remove tissue. This binary decision between removing and leaving tissue during surgery implies that the surgeon should be able to distinguish tumor from healthy tissue. In neurosurgery, current image-guidance approaches such as magnetic resonance imaging (MRI) combined with neuro-navigation offer a map as to where the tumor should be, but the only definitive method to characterize the tissue at stake is histopathology. While extremely valuable information is derived from this gold standard approach, it is limited to very few samples during surgery and is not practically used for the delineation of tumor margins. The development and implementation of faster, comprehensive and complementary approaches for tissue characterization are required to support surgical decision-making -an incremental and iterative process with tumor removed in multiple and often minute biopsies. The development of atmospheric pressure ionization sources makes it possible to analyze tissue specimens with little to no sample preparation. Here, we highlight the value of desorption electrospray ionization (DESI) as one of many available approaches for the analysis of surgical tissue. Twelve surgical samples resected from a patient during surgery were analyzed and diagnosed as glioblastoma (GBM) tumor or necrotic tissue by standard histopathology, and mass spectrometry results were further correlated to histopathology for critical validation of the approach. The use of a robust statistical approach reiterated results from the qualitative detection of potential biomarkers of these tissue types. The correlation of the MS and histopathology results to magnetic resonance images brings significant insight into tumor presentation that could not only serve to guide tumor resection, but that is worthy of more detailed studies on our understanding of tumor presentation on MRI.