Traditional Chinese medicines (TCMs) are getting more and more popular nowadays in the whole world for improving health condition of human beings as well as preventing and healing diseases. TCM is a multi-component system with components mostly unknown, and only a few compounds are responsible for the pharmaceutical and/or toxic effects. The large numbers of other components in the TCM make the screening and analysis of the bioactive components extremely difficult. So, separation and analysis of the desired chemical components in TCM are very important subjects for modernization research of TCM. Thus, many novel separation techniques with significant advantages over conventional methods were introduced and applied to separation and analysis of the chemical constituents in TCM. This review presents just a brief outline of the applications of different separation methods for the isolation and analysis of TCM constituents.