Glioblastoma (GBM) is a devastating brain tumor with limited treatment success, partly because in vitro models poorly mimic in vivo complexity. This study introduces a high-throughput 3D culture platform utilizing modular starPEG–glycosaminoglycan (GAG) hydrogels that enable independent control of extracellular matrix (ECM) cues: stiffness, cytokine affinity, matrix metalloproteinase-responsive remodeling, and cell adhesiveness via integrin-binding RGD peptides. This platform supports encapsulation of patient-derived GBM cells, recreates physiologically relevant tumor microenvironments in 384-well plates, and enables automated drug testing on primary cells. Transcriptomic analyses show that 3D cultures recapitulate primary and recurrent GBM programs- including hypoxia-, immune-, and ECM-regulatory pathways driving growth, invasion, and resistance, without externally imposed hypoxia. The platform's versatility extends to drug screening, where single and combinatorial treatments produce reproducible cytoskeletal and transcriptomic responses. Notably, the system revealed dose-dependent reductions in invasive filaments and spheroid architecture with 5-fluorouracil/uridine and carmustine, demonstrating its potential for optimizing combinatorial therapies. This 3D model surpasses 2D cultures, capturing tumor-specific molecular programs and offering a robust tool for translational research. Despite lacking vascular or immune components, its tunability, scalability, and clinical relevance make it a strong basis for advanced co-cultures. By delivering reliable, individualized therapeutic data within a short timeframe, this model holds transformative potential for personalized GBM treatment.