Microglia are non-neuronal cells, which reside in the central nervous system and are known to play an important role in health and disease. We investigated the lipidomic phenotypes of human naı̈ve and stimulated microglia-like cells by atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI). With lateral resolutions between 5 and 1.5 μm pixel size, we were able to chart lipid compositions of individual cells, enabling differentiation of cell lines and stimulation conditions. This allowed us to reveal local lipid heterogeneities in naı̈ve and lipopolysaccharide (LPS)-stimulated cells. We were able to identify individual cells with elevated triglyceride (TG) levels and could show that the number of these TG-enriched cells increased with LPS stimulation as a hallmark for a proinflammatory phenotype. Additionally, the observed local abundance alterations of specific phosphatidylinositols (PIs) indicate a cell specific regulation of the PI metabolism.