Polyphenols are among the most abundant and functionally diverse classes of secondary metabolites synthesized by plants. These organic compounds, characterized by multiple phenol structural units, play an essential role in plant defense systems, organoleptic quality, and medicinal bioactivity. Due to their potent antioxidant, anti-inflammatory, antimicrobial, and cardioprotective attributes, polyphenol quantification and profiling have become indispensable quality control parameters in the standardization of herbal medicines and botanical dietary supplements.

This review provides a systematic synthesis of the principal analytical methodologies currently utilized for assessing polyphenol content in raw plant materials and derived pharmaceutical formulations. Specifically, we examine three major analytical paradigms: colorimetric assays via the Folin–Ciocalteu (FC) reagent, high-performance liquid chromatography (HPLC) paired with spectrophotometric or electrochemical detectors, and liquid chromatography coupled with mass spectrometry (LC–MS). The fundamental principles, sensitivity, selectivity, operational costs, sample preparation protocols, and limitations of each technique are systematically critically analyzed and contrasted.

The colorimetric Folin–Ciocalteu method offers an accessible, cost-effective screening tool for total phenolic content (TPC), though it suffers from cross-reactivity with non-phenolic reducing compounds. Conversely, HPLC provides high resolution and precision for quantifying individual marker compounds, albeit requiring pure standard reference compounds and extensive chromatographic optimization. LC–MS represents the gold standard for structural elucidation, isomer differentiation, and high-throughput profiling of complex phenolic matrices without always requiring reference standards, though bounded by higher capital expenditure and operational complexity. Finally, this review highlights prospective trends in green extraction technologies, miniaturized analytical devices, and high-resolution mass spectrometry aimed at improving reliability, eco-friendliness, and precision in botanical quality control.

Keywords: Polyphenols, Folin–Ciocalteu Assay, HPLC, LC–MS, Quality Control.