Lipid alterations in the brain are well-documented in disease and aging, but our understanding of their pathogenic implications remains incomplete. Recent technological advances in assessing lipid profiles have enabled us to intricately examine the spatiotemporal variations in lipid compositions within the complex brain characterized by diverse cell types and intricate neural networks. In this study, we coupled time-of-flight secondary ion mass spectrometry (ToF-SIMS) to an amyotrophic lateral sclerosis (ALS) Drosophila model, for the first time, to elucidate changes in the lipid landscape and investigate their potential role in the disease process, serving as a methodological and analytical complement to our prior approach that utilized matrix-assisted laser desorption/ionization mass spectrometry. The expansion of G4C2 repeats in the C9orf72 gene is the most prevalent genetic factor in ALS. Our findings indicate that expressing these repeats in fly brains elevates the levels of fatty acids, diacylglycerols, and ceramides during the early stages (day 5) of disease progression, preceding motor dysfunction. Using RNAi-based genetic screening targeting lipid regulators, we found that reducing fatty acid transport protein 1 (FATP1) and Acyl-CoA-binding protein (ACBP) alleviates the retinal degeneration caused by G4C2 repeat expression and also markedly restores the G4C2-dependent alterations in lipid profiles. Significantly, the expression of FATP1 and ACBP is upregulated in G4C2-expressing flies, suggesting their contribution to lipid dysregulation. Collectively, our novel use of ToF-SIMS with the ALS Drosophila model, alongside methodological and analytical improvements, successfully identifies crucial lipids and related genetic factors in ALS pathogenesis.