Extracts of milk thistle (Silybum marianum, Asteraceae) have been recognized for centuries as remedies for liver and gallbladder disorders. The active constituents of milk thistle fruits are flavonolignans, collectively known as silymarin. Flavonolignans in S. marianum are structurally diverse, 23 constituents have been isolated from purple- and white-flowering variants. Flavonolignans have a broad spectrum of bioactivities and silymarin has been the subject of intensive research for its profound pharmacological activities. Silymarin is extracted from the seeds, commercialized in standardized form, and widely used in drugs and dietary supplements. The thorough analysis of silymarin, its constituents and silymarin-containing products has a key role in the quality control of milk thistle-based products. Due to the low concentration of analytes, especially pharmacological and pharmacokinetic studies require more and more selective and sensitive, advanced techniques. The objective of the present review is to summarize the recent advances in the chemical analysis of S. marianum extracts, including the chemical composition, isolation and identification of flavonolignans, sample preparation, and methods used for qualitative and quantitative analysis. Various analytical approaches have been surveyed, and their respective advantages and limits are discussed.