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Imaging mass spectrometry provides both chemical information and the spatial distribution of each analyte detected. Here it is demonstrated how imaging mass spectrometry of tissue at subcellular resolution can be achieved by combining the high spatial resolution of secondary ion mass spectrometry (SIMS) with the sample preparation protocols of matrix‐assisted laser desorption/ionization (MALDI). Despite mechanistic differences and sampling 10⁵ times less material, matrix‐enhanced (ME)‐SIMS of tissue samples yields similar results to MALDI (up to m/z 2500), in agreement with previous studies on standard compounds. In this regard ME‐SIMS represents an attractive alternative to polyatomic primary ions for increasing the molecular ion yield. ME‐SIMS of whole organs and thin sections of the cerebral ganglia of Lymnaea stagnalis demonstrate the advantages of ME‐SIMS for chemical imaging mass …