Glioblastoma (GBM) is an aggressive, World Health Organization grade 4 brain tumor with a poor prognosis, largely due to its complex, treatment-resistant microenvironment. To better model this environment for preclinical testing, we developed a three-dimensional (3D) biomimetic bioprinting platform using patient-derived GBM cells. Two hydrogels, alginate/gelatin (AlgGel; 1.5%/7.5%) and gelatin methacryloyl (10%), were evaluated for biocompatibility. GBM cells (GBM06, GBM14, and GBM15), transduced with iRFP-680 for viability tracking, were embedded in the hydrogels and printed. Tumor growth and viability were monitored for 28 days using fluorescence microscopy, complemented by electron microscopy (EM) for structural analysis. Drug response testing included temozolomide (10 µM) and the cyclin-dependent kinases 4/6 inhibitor abemaciclib (1 µM). Cell viability and extracellular vesicle release were quantified. Efficacy was further assessed in a co-culture with astrocytes. The AlgGel hydrogel supported superior long-term viability and growth. EM analysis of AlgGel scaffolds revealed preserved cellular architecture and adherence to the bioprinted extracellular matrix. Drug response assays confirmed findings previously observed in 2D and 3D cultures. Two cycles of abemaciclib reduced GBM cell viability in AlgGel scaffolds, accompanied by a significant decrease in extracellular vesicle secretion. Temozolomide, in contrast, did not significantly affect cell viability. The reduction in viability remained pronounced in co-culture with astrocytes, without compromising astrocyte viability. In this study, we present a 3D biomimetic bioprinting model that successfully mimics key aspects of the GBM microenvironment. This model demonstrates strong potential as a preclinical drug screening tool, enabling improved mechanistic insight into cell–matrix interactions that govern nutrient/metabolite diffusion and therapeutic responses.