Mass spectrometry provides multiple options for the direct characterization of tissue to support surgical decision-making, and provides significant insight in the development of drugs targeting tumors of the central nervous system (CNS). Using an array of mass spectrometry (MS) applications, we rapidly analyze specific tumor markers ranging from small metabolites to proteins from surgical tissue for rapid diagnosis and surgical guidance. With similar clinical protocols and in pre-clinical animal studies, we visualize drug and metabolites penetration in brain tumor tissue and correlate with tumor heterogeneity and response to support drug development.