Brain tumor heterogeneity poses significant challenges to their study and moreover to their treatment. A tool that could characterize a tumor specimen based on its cellular composition and response to treatment could assist the treating neuro-oncologist in determining the optimal treatment. We have developed a chip to culture dissociated cells in an organized array to categorize cell populations and subject them to targeted therapy. The chip was designed for analysis by optical and mass spectrometry imaging. The microwell array coupled to matrix assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) is used for the identification of clinical markers of response to targeted therapy. The new microwell array was validated for single-cell imaging by mass spectrometry based on correlation to fluorescence microscopy. Adherent NIH3T3 mouse fibroblasts expressing the enhanced GFP reporter gene and/or U87 cells stained with DAPI were incubated on microwell plates to allow their isolation at the bottom of each well. After cell fixation, the chip was imaged by fluorescence microscopy to determine the total number of wells occupied by cells, and the number of cells per well. The chips were coated with the CHCA matrix and analyzed by MALDI MSI. The statistical analyses of the MALDI MSI data revealed molecular signatures characteristic of each cell type, and mostly assigned to phospholipids. We also monitored the U87 cell response to a targeted inhibitor treatment to identify single-cell barcoding of drug response by MALDI MSI. The platform is further applied to the analysis of dissociated cells from surgical glioma specimens to characterize their heterogeneity and evaluate targeted treatment response. The proposed research has the potential to open ways for more comprehensive diagnostic approaches and to accompany them with direct evaluation of drug effect evaluation for the tailoring of brain cancer treatment.