Phenolic compounds, ubiquitous in plants, are of considerable interest and have received more and more attention in recent years due to their bioactive functions. Polyphenols are amongst the most desirable phytochemicals due to their antioxidant activity. These components are known as secondary plant metabolites and possess also antimicrobial, antiviral and anti-inflammatory properties along with their high antioxidant capacity. Many efforts have been made to provide a highly sensitive and selective analytical method for the determination and characterisation of polyphenols. The aim of this paper is to provide information on the most recent developments in the chemical investigation of polyphenols emphasising the extraction, separation and analysis of these compounds by chromatographic and spectral techniques.