AbstractDespite aggressive therapeutic regimens, glioblastoma (GBM) represents a deadly brain tumor with significant aggressiveness, radioresistance and chemoresistance, leading to dismal prognosis. Hypoxic microenvironment, which characterizes GBM, is associated with reduced therapeutic effectiveness. Moreover, current irradiation approaches are limited by uncertain tumor delineation and severe side effects that comprehensively lead to unsuccessful treatment and to a worsening of the quality of life of GBM patients. Proton beam offers the opportunity of reduced side effects and a depth-dose profile, which, unfortunately, are coupled with low relative biological effectiveness (RBE). The use of radiosensitizing agents, such as boron-containing molecules, enhances proton RBE and increases the effectiveness on proton beam-hit targets. We report a first preclinical evaluation of proton boron capture therapy (PBCT) in a preclinical model of GBM analyzed via μ-positron emission tomography/computed tomography (μPET-CT) assisted live imaging, finding a significant increased therapeutic effectiveness of PBCT versus proton coupled with an increased cell death and mitophagy. Our work supports PBCT and radiosensitizing agents as a scalable strategy to treat GBM exploiting ballistic advances of proton beam and increasing therapeutic effectiveness and quality of life in GBM patients.