This chapter presents a universal protein extraction protocol specifically designed for phenolic-rich legume tissues and optimized for proteomic analysis using liquid chromatography coupled with mass spectrometry (LC-MS/MS). The protocol includes a methanol: chloroform: water (MCW) extraction step that effectively removes secondary metabolites such as pigments, phenolic compounds, lipids, carbohydrates, and terpenes, resulting in cleaner protein samples. It is cost-effective, requires minimal tissue (less than 20 mg), and can identify over 2,000 proteins per sample. Additionally, this method offers enhanced proteome coverage, sensitivity, and quantitative accuracy compared to traditional 2D gel electrophoresis techniques.

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