Abstract: Mass spectrometry imaging(MSI)is a new analytical technique to study the composition and distribution of molecules or ions at tissue, cellular or subcellular level. It can detect analytes qualitatively and quantitatively on sample surface in situ without complex pre-treatment, mapping their spatial distribution. MSI technique is now emerging with its advantages in pharmaceutical science. Principle, sample preparation and ionization techniques concerning MSI are introduced in this review. Its application in drug metabolism study, pharmacodynamic and toxicological evaluation, pharmacoproteomics exploration, targeted drug screening and solid preparations investigation are also reviewed.