This study employs a multi-omics approach to investigate the biochemical impact of AFG3L2 mutations in immortalized lymphoblastoid cell lines derived from a SPAX5 patient. Our proteomic analysis revealed significant dysregulation in proteins involved in mitochondrial function, cytoskeletal integrity, and cellular metabolism. Specifically, we observed disruptions in mitochondrial dynamics and calcium homeostasis, characterized by the downregulation of critical mitochondrial proteins such as COX11 and NFU1, and upregulation of key regulatory proteins like PRKCB and KCTD12. Consistent with this, metabolomic profiling identified a marked reduction in acetyl-CoA levels, indicating impaired TCA cycle activity and potential energy deficits. Lipidomic analysis highlighted substantial alterations in lipid composition, with significant decreases in sphingomyelins, phosphatidylethanolamine, and phosphatidylcholine, reflecting disruptions in lipid metabolism and membrane integrity. Our comprehensive investigation into AFG3L2 deficiency elucidates a pathophysiology extending beyond mitochondrial proteostasis, implicating a wide array of cellular processes. The findings reveal substantial cellular disturbances at multiple levels, contributing to neurodegeneration through disrupted mitochondrial calcium homeostasis, cytoskeletal integrity, and altered metabolic and lipid homeostasis. This study underscores the complexity of SPAX5 pathophysiology and the importance of multi-omics approaches in developing effective strategies to address the impact of AFG3L2 deficiency. Our data also highlight the value of immortalized lymphoblastoid cells as a tool for pre-clinical testing and research, offering a detailed biochemical fingerprint that enhances our understanding of SPAX5 and identifies potential areas for further investigation.