Matrix-assisted laser desorption/ionization (MALDI) and mass spectrometry (MS) imaging are advanced technologies capable of revealing the spatial distribution of different molecules - e.g., drugs and their metabolites, endogenous lipids and complex peptides/proteins - directly in tissue specimens at the same time. Information obtained regarding tissues by MALDI profiling/imaging analysis can be correlated with other MS-based techniques, auxiliary imaging technologies and routine immunohistochemical stainings. In this review we describe the MALDI profiling/imaging technologies, providing examples of their application in kidney research.