The project aimed at proteomic profiling of patient-derived glioblastoma organoids from fragment-based (CUT) and dissociation-based (DIS) approaches of organoid generation. Organoids from multiple patient tumors were cultured under growth factor–free or growth factor–supplemented conditions and compared with matched primary tissue. Both protocols (CUT and DIS) produced technically robust glioblastoma organoids when maintained in their native media. CUT organoids matched the reproducibility of DIS cultures while preserving a broader extracellular matrix repertoire and networks linked to collagen assembly, vascular support, and cell–matrix signaling. DIS cultures were biased toward exogenous basement membrane components and proliferative, growth factor–responsive states. Across tumors, CUT organoids consistently showed greater proteomic similarity to matched primary tissue, retaining neural, glial, stromal, and extracellular features largely absent from DIS models.