Diffuse midline glioma is one of the most difficult pediatric cancers to treat. This study investigated the feasibility of 13C magnetic resonance metabolic imaging of hyperpolarized [1-13C]pyruvate for monitoring response to novel therapies in diffuse midline glioma. Treatment with panobinostat was associated with a reduction in hyperpolarized lactate and a reduced LDHA activity in an in vitro experiment. Radiotherapy led to a reduction in the ratio of lactate to pyruvate in rats bearing diffuse midline glioma. The results suggest that hyperpolarized 13C metabolic imaging may provide an early noninvasive biomarker to monitor therapy response in diffuse midline glioma.