For a study, researchers sought to test whether compromising HR by targeting HSP90, a molecular chaperone required for the function of key HR proteins would sensitize high-grade gliomas to chemoradiation in vitro and in vivo using analepsis, a long-acting, brain-penetrant HSP90 inhibitor. Onalespib was tested in vitro in GSCs and in vivo in zebrafish and mouse intracranial glioma xenograft models to deplete HR client proteins, impair HR repair capacity, and sensitize HR glioblastoma (GBM) to chemoradiation. In GSCs and ex vivo organotypic human glioma slice cultures, the effects of HSP90 inhibition on the transcriptome and cytoplasmic proteins were studied. Onalespib treatment depleted CHK1 and RAD51, 2 key proteins in the HR pathway, and reduced HR repair, making GSCs more susceptible to radiation and temozolomide (TMZ). HSP90 inhibition reprogrammed the transcriptome of GSCs and altered the expression of cytoplasmic proteins, including known and novel GSC-related client proteins. In a zebrafish and a mouse xenograft model of GBM, combining onalespib with radiation and TMZ outlived the standard of care (radiation and TMZ) or onalespib alone. The findings showed that inhibiting HSP90 sensitizes GSCs to radiation and chemotherapy and increases survival in zebrafish and mouse intracranial models of GBM. These findings provided a preclinical rationale for testing HSP90 inhibitors in combination with chemoradiation in GBM patients.