Abstract: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), as a label-free imaging technique with high coverage and sensitivity is widely used for visualizing the spatial distribution of proteins, peptides and small metabolites in tissues. With the development of MALDI technique, MALDI-MSI is also employed to monitor the spatial distribution of phytochemical constituents of medicinal plants. In this review, we first briefly introduce MALDI-MSI technique, and we focus on its application in the spatial distribution and accumulation of secondary metabolites in medicinal plants. The ultimate advantage of using MALDI-MSI for spatial distribution analysis at the molecular level, offers crucial evidence of synthesis, transfer and accumulation of bioactive molecules in medicinal plants.