Glioblastoma multiforme (GBM) is the most common and most aggressive type of primary brain tumor. Identification of new therapeutic regimens is urgently needed. A major challenge remains the development of a relevant in vitro model system with the necessary capacity and flexibility to profile compounds. The authors have developed and characterized a 3D culture system of brain cells (brain Hi-Spot) where GBM-derived cells can be incorporated (GBM/brain Hi-Spot). Immuno-fluorescence and electrophysiological recordings demonstrate that brain Hi-Spots recapitulate many features of brain tissue. Within this tissue, GBM-derived cell growth is monitored using a fluorescence assay. GBM-derived cells growing in Hi-Spots form tumor nodules that display properties of GBM such as 5-Ala positive staining, an acidic environment, and tumor-surrounding astrocyte activation. Temozolomide inhibits GBM growth in brain Hi-Spots, but it is not effective in 2D cultures. Other chemotherapeutics that have proven to be inefficient in GBM treatment display low activity against GBM-derived cells growing in brain Hi-Spots in comparison to their activity against GBM 2D cultures. These findings suggest that GBM/brain Hi-Spots represent a simple system to culture cells derived from brain tumors in an orthotopic environment in vitro and that the system is reliable to test GBM targeting compounds.