Background: GBM is the most common brain tumor in humans and has a dismal prognosis. Although antiangiogenic therapy (bevacizumab) is an option for GBM, there is still unmet need to understand tumor pathophysiology and predictive biomarkers. We built a tissue based multiplexed immunofluorescent assays and developed algorithms to identify and quantify tumor vasculature, that enabled quantification, visualization, and colocalization of multiple protein in surrounding tumor cells at single cell and subcellular levels. This assay provides unique opportunity to explore tumor heterogeneity of tissue morphology and their relationships to vasculature, and is a novel tool for biomarker and treatment discovery. Methods: Tissue micro arrays (TMAs) were constructed from 141 GBM patients. Fluorescent dye labeled antibodies against 18 biomarkers were sequentially applied on single sections of these TMAs. Metrics were built to identify vessels, quantify distance of tumor cells to vessels, and analyze expression profiles of biomarkers, including signaling molecules in EGFR, PI3K/AKT, TGF-beta pathways, and hypoxia marker Glut1, in proximity to blood vessels. Results: CD31 was successfully used to identify blood vessels in GMB. Vessel segmentation and quantification were performed on all of the images. Biomarker profiling in the context of blood vessels demonstrated different patterns in close proximity to vessels, with some biomarkers showing increased levels (e.g. SMA, EGFR, pS6), some showing decreased levels (e.g. p4EBP), and others remain the same (FOXO3a, S6). Quantification of biomarkers showed heterogeneous expression within the same sample and across the cohort. In addition, co-localization of the above markers was visualized and demonstrated on single cell and subcellular levels. Conclusions: We were able to use a novel fluorescent multiplexing technology (MultiOmyx) to study GBM biology. This technology allowed the simultaneous analyses of multiple biomarkers of GBM, and provides new insights on the relationship of markers to each other, tumor heterogeneity and angiogenesis.