MALDI (Matrix-Assisted Laser Desorption/Ionization) Imaging mass spectrometry is a powerful new method for analyzing the spatial distribution of molecules in tissues. Several different classes of cellular constituents such as proteins, peptides, lipids, and small molecules can be analyzed in situ while maintaining the morphological integrity of the tissue. This allows a correlation of the morphology with the previously acquired molecular patterns. By this, specific molecules can be clearly assigned to their cellular origin. Here, we will present a protocol for the analysis of proteins in tissues which are either native or alcohol-fixed and paraffin-embedded.