Drosophila melanogaster (fruit fly) has a relatively simple nervous system but possesses high order brain functions similar to humans. Therefore, it has been used as a commonmodel system inbiological studies, particularly drug addiction. Here, the spatial distribution of biomolecules in the brain of the fly was studied using time-of-flight SIMS. Fly brains were analyzed frozen to prevent molecular redistribution prior to analysis. Different molecules were found to distribute differently in the tissue, particularly the eye pigments, diacylglycerides, and phospholipids, and this is expected to be driven by their biological functions in the brain. Correlations in the localization of these moleculeswere also observed using principal components analysis of image data, and this was used to identify peaks for further analysis. Furthermore, consecutive analyses following 10 keV Ar2500 + sputtering showed that different biomolecules respond differently to Ar2500 + sputtering. Significant changes in signal intensities between consecutive analyses were observed for high mass molecules including lipids.