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Matrix-assisted laser desorption/ionization (MALDI) imaging is rapidly gaining importance in the -biomarkers field because it is able to detect several analytes at the same time and to assign to each one of them not only an m/z value but also spatial distribution. Here we present the detailed description of sample preparation for protein and peptide MALDI imaging assays. This chapter describes the microtomy performed in a cryostat to produce tissue slides mounted onto glass slides suitable for MALDI. Sample preparation will include matrix coating procedure with a sensor-controlled aerosol. Finally, we will show some examples of how data can be visualized to suit the purposes of the research.