Abstract: Drugs with more than one chiral center are usually involved with different stereo-specific isomers and big difference in bioactivity due to different stereo-chemical structure, its separation and analysis is still a great challenge. There are lots of reports about enantioseparation of racemate with one chiral center, however, only a small fraction number of papers about stereoselective separation of racemate with multiple chiral center are available. In this paper, stereoselective separation of racemate with more than one chiral center by various chromatographic techniques, including HPLC, GC, capillary electrophoresis, supercritical fluid chromatography and countercurrent chromatography are reviewed.