Polyphenols are widespread in vegetables and fruits. They can play an important role in human diet and health, and they influence the sensorial properties of many foods, and act as natural antioxidants. This study was conducted using HPLC/DAD, tyrosinase biosensor, and differential pulse voltammetry (DPV) analyses to detect polyphenolic compounds in natural complex matrices. The analyses were applied to a series of both standards and natural extracts derived from grape, olives, and green tea. The pure compounds include phenolic acids, flavones, flavonols, catechins, tannins, and oleuropein. HPLC/DAD, DPV, and the biosensor approach were used as independent analytical techniques. Bare graphite screen-printed electrodes were employed in DPV and in the biosensor analysis. The most accurate data were obtained by HPLC/DAD analysis, while the DPV approach using screen-printed electrodes could represent a quick screening method for the determination of polyphenols in natural extracts. Use of the biosensor for the analysis of complex matrices needs further study in order to improve its performance.