Abstract: Matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) has been developed rapidly in recent years and has been widely used in many fields. Although MALDI MSI can obtain multiple imaging data of different compounds in one experiment, the imaging analysis of small molecules with low abundance, low ionization efficiency, and matrix interference is still very challenging. In order to improve the signal response of target molecules in MALDI MSI, it is necessary to apply the on-tissue derivatization to MALDI MSI. In recent years, the combination of derivatization and mass spectrometry imaging has shown excellent results for the analysis of target molecules containing reactive groups such as carboxyl, amino, and aldehyde groups. This review aims to introduce the application of on-tissue derivatization on MALDI MSI, including developed derivatization reagents and pretreatment methods for derivatization, the selection of matrix and the reagent coating device. Finally, the perspectives for derivatization in mass spectrometry imaging are outlooked.