This chapter presents a detailed protocol for analyzing the chemical composition of winery by-products, such as grape pomace, seeds, and stems, with the aim of facilitating their valorization into high-value products. It outlines the comprehensive methods for extracting polyphenols, determining total phenolic and flavonoid contents, and assessing antioxidant activities. It includes step-by-step instructions for using high-performance liquid chromatography (HPLC) and spectrophotometric techniques to accurately identify and quantify bioactive compounds. The extraction process utilizes solvents like methanol, ethanol, and acetone, while the antioxidant assays employ 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging tests. The calibration curves for polyphenols and antioxidant standards are provided, ensuring precise quantification. The manual emphasizes the importance of using analytical-grade reagents and maintaining instrument accuracy to obtain reliable results.