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Some ionic liquids have good potential to play the role of anti-solvents in the crystallization of natural active compounds after their initial extraction. In order to prove it and explore suitable candidates, a novel separation method of berberine hydrochloride was developed based on benzothiazolium ionic liquid induced crystallization in this study. Ionic liquids [C 6 Bth]PF 6 and [HBth]BF 4 were selected to conduct the separation process. The effects of various conditions on IL performance were investigated, under which the separation efficiency could achieve above 90% just by using a small amount of these ionic liquids. Moreover, the separation process was greatly simplified due to the different solubility of ionic liquids and berberine hydrochloride, so the further purification of target compounds and recycling process of the ILs could be avoided. The whole process is very easy to be scaled up and has been practiced for actual herbal sample. • Benzothiazolium ILs were reported in the crystallization for the first time. • A new separation method of IL-induced crystallization was developed. • High separation efficiency could be achieved through simple steps. • Conditions were investigated detailedly and practiced for herbal sample. [ABSTRACT FROM AUTHOR]