The discovery of new therapies and understanding of disease mechanisms all rely on efficient cell culturing techniques, either through in vivo animal models or two-dimensional (2D) in vitro cell models. However, 2D cell culture models have proved unreliable because of their inability to recreate the dynamic interactions between cells and the extracellular matrix (ECM), while animal studies can be expensive and time-consuming. Three-dimensional (3D) cell culture models recapitulate the in vivo environment, allow for cell-cell and cell-matrix interactions, and provide a platform for representation of the therapeutic responses of several diseases, which can then be used to determine their effectiveness. Glioblastoma (GBM), a lethal and aggressive primary malignant brain tumor, is one such disease for which treatment development will require physiologically relevant models that recreate the interaction between the ECM environment and the cancer cells. These interactions aid cancer cell survival and proliferation and contribute to GBM’s ability to acquire resistance to chemotherapeutic drugs. 3D biomaterial models can be used to represent the brain and brain tumor ECM and study tumor cell-ECM interactions. However, while it is known that cells cultured in 3D result in more physiologically relevant responses to treatments compared to cells cultured in 2D, differences in the chemical, physical, and mechanical properties of biomaterials used to fabricate 3D matrices can result in varying cell responses. Here, we compared responses of patient-derived GBM cells cultured in various 3D hydrogel matrices to the alkylating chemotherapy, temozolomide. Specifically, we assessed 3D cultures in two commercially available hydrogel platforms, HyStem and Matrigel, and two “in-house” hyaluronic acid (HA)-based hydrogels crosslinked using either a thiol-ene photo-click chemistry or a kinetic Michael-addition chemistry. In general, we found that the HyStem scaffolds were incapable of providing an appropriate environment for GBM cell adhesion and proliferation. In the Matrigel condition, we determined that temozolomide elicited a response that is not physiologically relevant. The scaffolds crosslinked with the Michael-type addition chemistry and thiol-ene photo-click chemistry showed similar cell behavior to temozolomide, both of which were more indicative of what has been observed in vivo. The two “in-house”, bioengineered matrices, which better approximate the brain tumor ECM and GBM cell interactions and lead to therapeutic responses reflected in clinical outcomes, can be used in future research to further explore the dynamics of developing resistance to chemotherapy drugs in vitro and screen new potential therapeutics.