MALDI-TOF MS is a powerful tool to analyze biomolecules,owingto its soft ionization nature that generally results in simple spectraof singly charged ions. Implementation of the technology in the imagingmode provides a means to spatially map analytes in situ. Recently,a new matrix, DBDA (N1,N4-dibenzylidenebenzene-1,4-diamine)was reported to facilitate the ionization of free fatty acids in negative ion mode. Building on this finding, we sought to implementDBDA for MALDI mass spectrometry imaging studies in brain tissue andsuccessfully map oleic acid, palmitic acid, stearic acid, docosahexaenoicacid, and arachidonic acid using mouse brain sections. Moreover, wehypothesized that DBDA would provide superior ionization for sulfatides,a class of sulfolipids with multiple biological functions. Herein,we also demonstrate that DBDA is ideal for MALDI mass spectrometryimaging of fatty acids and sulfatides in brain tissue sections. Additionally,we show enhanced ionization of sulfatides using DBDA compared withthree different traditionally used MALDI matrices. Together theseresults provide new opportunities for studies to measure sulfatidesby MALDI-TOF MS.