Plants contain phenolic compounds, which are of great importance and have drawn increasing attention recently because of their bioactive properties. Polyphenols’ antioxidant properties make them one of the most sought-after phytochemicals. These substances might be free or matrix-bound, monomers or highly polymerized structures, aglycones or glycosides, or both. Additionally, their stability varies significantly, and there is uneven distribution within the plant. Thus, a single standard technique for isolating and characterizing polyphenols cannot be recommended. Therefore, methods need to be tuned according to the study’s objective, sample type, and target analytes. This chapter has discussed the general protocols for preparing samples, isolation, and characterization approaches that range from conventional chromatographic procedures to more modern modified approaches.