Flavonoids, a diverse class of polyphenolic compounds found abundantly in plant sources, are increasingly recognized for their therapeutic potential, particularly in antioxidant, anti-inflammatory, anticancer, and cardioprotective applications. Realizing their full pharmacological and commercial value, however, requires robust and reproducible methods for their isolation and purification from complex plant matrices. This chapter will cover a comprehensive overview of the current methodologies and emerging innovations in the extraction, purification, and characterization of flavonoids. The extraction techniques will include conventional (e.g., maceration, Soxhlet) and advanced green extraction techniques like ultrasound-assisted and microwave-assisted. The chapter also explores purification strategies, including liquid–liquid extraction, column chromatography, membrane filtration, and preparative HPLC, while highlighting analytical approaches such as UV–Vis, HPLC, LC–MS, and NMR for structural elucidation and quantification. Overall, the chapter serves as a foundational guide for researchers and industry professionals aiming to develop efficient, eco-friendly, and scalable workflows for flavonoid recovery and standardization.