Abstract: Mass spectrometry (MS) is a kind of analytical method widely used in chemistry, life science, medicine and other fields. This method can provide information on molecular weight and is very helpful to structure analysis of unknown substance. It has advantages of high resolution, informative, less dosage of samples, sensitive, rapid, etc. MS plays an important role in the determination of accurate molecular weight of organic compounds, its structural analysis and the reaction mechanism research. As chiral drugs separation is a hot research topic in the field of medicine, MS has also been gradually applied to the distinction between the chiral impurities in recent years. With the continuous development of MS technology, it has obtained more and more attention in the analysis of chiral drug. This paper mainly reviewed the application and development history of MS in drug stereoisomer analysis of late years. The primary chiral MS methods in the current state come down to four categories, such as host-guest (H-G) method, ion/molecule (equilibrium) reactions, collision-induced dissociation (CID) and ion mobility mass spectrometry (IMS). The collision-induced dissociation (CID) of diastereomeric complex can be subdivided into three types for kinetic method (KM), fixed ligand kinetic method and chiral recognition ratio method (CR). All these MS methods were introduced considering their stereoisomer analysis principle and characteristics in this review. Examples were taken to make detailed instructions of the applications and advantages of the KM and IMS, which are two most popular MS methods said above. It has been widely accepted that mass spectrometry techniques have obvious advantages in separation and quantitative determination of stereoisomers owing to their high speed, accuracy and sensitivity. In conclusion, MS does not need complicated sample preparation and expensive chiral column when used to the optical impurities separation of chiral drugs, which greatly enhances the efficiency and accuracy in analysis of drug stereoisomers. The development of the mass spectrometry techniques would greatly promote the cutting-edge investigation and analysis of chiral impurities separation of drugs with multichiral centers and complicated conformation (such as polysaccharide, peptide) in the future.