Treatment resistance and recurrence continue to define the clinical landscape of glioblastoma (GBM), yet their study is limited by traditional in vitro frameworks that fail to capture long-term treatment dynamics and tumor evolution. Here we present GliaMimic, a longitudinal in vitro platform incorporating irradiation and multi-dose temozolomide (TMZ). Using patient-derived organoids (PDOs) and cell line-derived spheroids, tumor progression and treatment response were monitored non-invasively over four weeks. Substantial declines in metabolic activity and viability at clinically relevant TMZ concentrations (≤ 10 µM) emerge only after prolonged exposure, whereas short-term assays captured effects only at supraphysiological doses. Notably, PDOs, patient-derived spheroids, and their cell line-derived counterparts exhibited distinct patterns of treatment response and tumor progression, underscoring the importance of model selection in preclinical studies. Following treatment cessation, the platform captured distinct, patient-specific post-treatment tumor behaviors with persistent viable and metabolically active populations across all models with more pronounced changes in PDOs. By moving beyond static molecular diagnostics, GliaMimic provides a longitudinal in vitro treatment evaluation platform for preclinical testing.