Stereoisomers are a common form in natural products，and the pharmacological activities of isomers with different configurations are various. Efficient separation and analysis of stereoisomers is one of the urgent problems for the study of pharmacodynamic substances of natural products. In order to comprehensively and systematically review the separation and analysis methods of stereoisomers，this review used online databases PubMed，Web of Science，Google Scholar，ACS，CNKI，and others to comprehensive search for the separation and analysis of the isomeric，and systematically summaries the advantages，disadvantages and scope of application. The results showed that the separation methods of stereoisomers mainly included high performance liquid chromatography，two-dimensional liquid chromatography，supercritical fluid chromatography，gas chromatography and capillary electrophoresis. In addition，non-chromatographic methods such as preferential crystallization，membrane separation and kinetic splitting can also be used for stereoisomers splitting. Commonly used stereoisomer resolution methods include conventional mass spectrometry，ion mobility mass spectrometry，nuclear magnetic resonance techniques，single-crystal X-ray diffraction and spectroscopy. The study of common stereoisomer separation and analysis methods is summarized with a view to laying a foundation for the research and application of stereoisomers in natural products.