Metastatic glioblastoma multiforme (GBM) is known for its dismal prognosis due to the dissemination of single cells throughout the brain parenchyma and along white matter tracts, resulting in heightened resistance to therapies. Understanding the intricate relationship between cell migration, physical confinement, and chemotherapeutic resistance in GBM is imperative for advancing treatment strategies. In this study, we employed G55, a representative migratory GBM cell line, to investigate this phenomenon. We generated three distinct cell populations: (1) cells migrating without confinement, assessed via the Scratch assay; (2) cells migrating a short distance (10 μm) under confinement, examined through the Transwell assay; and (3) cells migrating long distances (>100 μm) under confinement, studied using the Microchannel assay. Comparative analyses of protein expression profiles and chemotherapy sensitivity among these groups revealed that migration combined with physical confinement plays a pivotal role in augmenting chemotherapeutic resistance in interstitial invasive cancer cells. Moreover, we demonstrate the utility of the microchannel device, which facilitates controlled cell migration under physical confinement, as an effective in vitro tool for investigating metastatic cancer and associated treatment resistance. This study sheds light on the mechanisms underlying GBM progression and highlights potential avenues for therapeutic intervention.