Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)-profiling and imaging mass spectrometry (MSI) are promising technologies for measuring hundreds of different molecules directly on tissues. For instance, small molecules, drugs and their metabolites, endogenous lipids, carbohydrates and complex peptides/proteins can be measured at the same time. In the most advanced instruments, it is achieved without significant disruption of sample integrity. MSI is a unique approach for assessing the spatial distribution of molecules using graphical multidimensional maps of their constituent analytes, which may for instance be correlated with histopathological alterations in patient tissues. MALDI-TOF-MSI technology has been implemented in hospitals of several countries, where it is routinely used for quick pathogen(s) identification, a task formerly accomplished by laborious and expensive DNA/RNA-based PCR (polymerase chain reaction) screening.In this chapter, we describe how MSI is performed, what is required from the researcher, the instrument vendors and finally what can be achieved with MSI. We restrict our descriptions only to MALDI-MSI although several other MS techniques of ionization can easily be linked to MSI.