Imaging mass spectrometry (IMS) has emerged as an important imaging modality because of its broad non-specific nature for molecular detection from highly complex samples. Within this broad field, new sub-categories of technologies have been developed incorporating many different molecular ionization processes. This article will focus on one of the major ionization processes, matrix-assisted laser desorption ionization (MALDI). IMS provides a critically important technology that brings new insight into complex biological samples and opens the door to new discoveries. Applications range widely, from fundamental studies in biology to specific clinical issues, all addressing the need to understand molecular spatial distributions at the tissue and cellular levels.