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HIGHLIGHTSClassification and general configuration of on‐line 2DLC are introduced.Applications of LC‐LC and LC × LC on chemical analysis of herbs are summarized.Future advance in on‐line 2DLC applied to chemical analysis of herbs is predicted. ABSTRACT Herbal medicines are complicated chemical systems containing hundreds of small molecules of various polarities, structural types, and contents. Thus far, the chromatographic separation of herbal extracts is still a big challenge. Two‐dimensional liquid chromatography (2DLC) has become an attractive separation tool in the past few years. Particularly, a lot of attention has been paid to on‐line 2DLC. In this review, we aim to give an overview on applications of on‐line 2DLC in the chemical analysis of herbal medicines since 2010. Firstly, classification and general configurations of on‐line 2DLC were briefly introduced. Then, we summarized main applications in herbal medicines of heart‐cutting 2DLC (LC‐LC), comprehensive 2DLC (LC × LC), and their combinations, with emphasis on LC × LC. Mass spectrometry is the most popular detector coupled with 2DLC, which allows sensitive and accurate structural characterization of herbal compounds. Finally, future developments in on‐line 2DLC techniques were also discussed.