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Through pharmacological activity research, an increasing number of natural products and their derivatives are being recognized for their therapeutic value. In recent years, studies have been conducted on Corydalis yanhusuo W.T. Wang, a valuable medicinal herb listed in the Chinese Pharmacopoeia. Protopine, one of its components, has also become a research hotspot. To illustrate the identification, metabolism, and broad pharmacological activity of protopine and the botanical preparations containing it for further scientific studies and clinical applications, an in-depth and detailed review of protopine is required. We collected data on the identification and quantification, metabolism and pharmacokinetics, pharmacological activities, and botanical preparations of protopine from 1986 to 2021 from the PubMed database using “protopine” as a keyword. It has been shown that protopine as an active ingredient of many botanical preparations can be rapidly screened and quantified by a large number of methods (such as the LC-ESI-MS/MS and the TLC/GC-MS), and the possible metabolic pathways of protopine in vivo have been proposed. In addition, protopine possesses a wide range of pharmacological activities such as anti-inflammatory, anti-platelet aggregation, anti-cancer, analgesic, vasodilatory, anticholinesterase, anti-addictive, anticonvulsant, antipathogenic, antioxidant, hepatoprotective, neuroprotective, and cytotoxic and anti-proliferative activities. In this paper, the identification and quantification, metabolism and pharmacokinetics, pharmacological activities, and botanical preparations of protopine are reviewed in detail to lay a foundation …