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Imaging using MS has the potential to deliver highly parallel, multiplexed data on the specific localization of molecular ions in tissue samples directly, and to measure and map the variations of these ions during development and disease progression or treatment. There is an intrinsic potential to be able to identify the biomarkers in the same experiment, or by relatively simple extension of the technique. Unlike many other imaging techniques, no a priori knowledge of the markers being sought is necessary. This review concentrates on the use of MALDI‐MS for MS imaging (MSI) of proteins and peptides, with an emphasis on mammalian tissue. We discuss the methodologies used, their potential limitations, overall experimental considerations and progress that has been made towards establishing MALDI‐MSI as a routine technique for the spatially resolved measurement of peptides and proteins. As well as …