Glioblastoma (GBM) is a highly aggressive brain tumor characterized by strong resistance to both chemotherapeutic and targeted therapeutic interventions. Our previous studies demonstrated that pharmacological inhibition of Focal Adhesion Kinase (FAK) and Proline-rich Tyrosine Kinase 2 (Pyk2) signaling in mouse glioma models significantly reduces tumor growth but results in only a modest increase in overall survival, suggesting the development of compensatory resistance mechanisms. Additionally, compensatory activation of the MEK/ERK signaling pathway was observed following the inhibition of FAK/Pyk2. We therefore hypothesized that concurrent targeting of FAK/Pyk2 and MEK/ERK signaling pathways could overcome MEK/ERK activation and enhance the therapeutic efficacy of FAK/Pyk2 inhibition in GBM. Western blot analysis of primary human GBM cell lines and the GL261 glioma model in C57BL/6 mice revealed increased phosphorylation of MEK and ERK following treatment with the dual Pyk2/FAK inhibitor defactinib (5 µM in vitro; 50 mg/kg in vivo) for 72 hours. Combinatorial treatment with the MEK/ERK inhibitor avutometinib (1 µM in vitro; 0.3 mg/kg in vivo) abolished this compensatory activation, resulting in a 10-fold reduction in ERK phosphorylation in vitro and a 3-fold reduction in vivo. Cell cycle analysis demonstrated an accumulation of cells in the G2/M phase and induction of apoptosis following 72-hour treatment with defactinib, indicating cell cycle arrest. In contrast, avutometinib treatment for 72 hours resulted in cell accumulation in the G0 phase, accompanied by a decrease in cyclin D1 expression, as confirmed by Western blot analysis, consistent with a cytostatic effect. Combined treatment induced cell cycle arrest at both the mitotic (G2/M) and G0 phases, reflecting the additive effects of both inhibitors. Cell viability assays supported these findings, showing that defactinib and avutometinib monotherapies induced approximately 50% and 20% cell death, respectively, together with 60% and 25% reductions in live cell populations compared to untreated controls. In conclusion, combined inhibition of Pyk2/FAK and MEK/ERK signaling produced an additive anti-tumor effect in glioma cells, suppressing compensatory MEK/ERK activation and enhancing growth inhibition.