Abstract <p>Yerba mate (<i>Ilex paraguariensis</i>) is a native South American plant widely recognized for its high content of polyphenols, particularly caffeoylquinic acids. During the industrial processing of yerba mate, by-products such as discarded leaves and branches are generated, providing low-cost alternatives for polyphenol extraction. The aim of this study was to validate a high-performance liquid chromatography method with diode array detection for the determination of the major polyphenols in yerba mate by-products, specifically six caffeoylquinic acids and the flavonoid rutin. The method was validated by assessing its linearity, detection and quantification limits, and repeatability, confirming its suitability for the analysis of yerba mate by-products extracts. The validated method was applied to the analysis of by-products from different yerba mate manufacturers. Quantitative analysis revealed significant differences in polyphenolic profiles and concentrations between leaves and branches. In leaf residue extracts, 3-caffeoylquinic acid was the most abundant polyphenol, whereas 5-caffeoylquinic acid was predominant in branches. These findings highlight the potential of yerba mate processing residues as low-cost sources of polyphenols, supporting the valorization of these by-products.</p>