Glioblastoma (GBM) is a devastating cancer of the brain with an extremely poor prognosis. Novel in-vitro methods for the assessment of cancer response to drugs and radiation are being developed. Compared to two-dimensional cell culture, three-dimensional cellular microenvironments provide a model closer to the in-vivo situation. In the context of cancer treatment, while X-Ray radiotherapy and chemotherapy remain the current standard, Proton beam therapy is an alternative with a compelling biological and medical rationale. It has been shown to reduce the damage to healthy tissue with superior targeting abilities. In this report, a novel 3D engineered scaffold is designed and fabricated by two-photon polymerization (2PP). 2PP provides high-resolution and reproducible scaffolds that are used to compare the response of cultured U251 cell line to Proton Beam irradiation. The cells are cultured on two- and three-dimensional scaffolds simultaneously for response comparison. Gamma-H2A.X is the marker used to identify the damage induced in the cells by the proton beams. The results show a higher DNA double-strand breakage in 2D cells as compared to those cultured in 3D. Differences in morphologies and proliferation are also observed. The discrepancy in proton radiation response could indicate a difference in the radioresistance of the cells or a difference in the rate of repair kinetics between 2D and 3D cells. Thus, these biomimetic engineered 3D scaffolds can be used to routinely assess the effects of proton therapy on tridimensional GBM cell networks. These scaffolds have also been proved to be effective with evaluation methods such as immunofluorescence and scanning electron microscopy.