Multiomics analysis of single tissue sections using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) provides comprehensive molecular insights. However, optimizing tissue sample preparation for MALDI-MSI to achieve high sensitivity and reproducibility for various biomolecules, such as lipids, N-glycans, and tryptic peptides, presents a significant challenge. This study introduces a robust and reproducible protocol for the comprehensive sequential analysis of the latter molecules using MALDI-MSI in fresh-frozen rodent brain tissue samples. The optimization process involved testing multiple organic solvents, which identified serial washing in ice-cold methanol, followed by chloroform as optimal for N-glycan analysis. Integrating this optimized protocol into MALDI-MSI workflows enabled comprehensive sequential analysis of lipids (in dual polarity mode), N-glycans, and tryptic peptides within the same tissue sections, enhancing both the efficiency and reliability. Validation across diverse rodent brain tissue samples confirmed the protocol's robustness and versatility. The optimized methodology was subsequently applied to a transgenic Alzheimer's disease (AD) mouse model (tgArcSwe) as a proof of concept. In the AD model, significant molecular alterations were observed in various sphingolipid and glycerophospholipid species, as well as in biantennary and GlcNAc-bisecting N-glycans, particularly in the cerebral cortex. These region-specific alterations are potentially associated with amyloid-beta (A beta) plaque accumulation, which may contribute to cognitive and memory impairments. The proposed standardized methodology represents a significant advancement in neurobiological research, providing valuable insights into disease mechanisms and laying the foundation for potential preclinical applications. It could aid the development of diagnostic biomarkers and targeted therapies for AD and other neurodegenerative diseases, such as Parkinson's disease.