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A new analytical method (liquid chromatography-antioxidant, LC-AOx) was used that is intended to separate beer polyphenols and to determine the potential antioxidant activity of these constituents after they were allowed to react online with a buffered solution of the radical cation 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(•+)). Using the LC-AOx method, it was possible to demonstrate that the extent of the antioxidant activity was very much dependent on the phenolic compound considered. The method was also applied to the analysis of beer extracts and allowed the evaluation of their antioxidant activity at different steps of beer processing: brewing, boiling, and fermentation. This study showed that the total antioxidant activity remained unchanged throughout beer processing, as opposed to the polyphenolic content, which showed a 3-fold increase. Hopping and fermentation steps were the main causes of this increase. However, the increase measured after fermentation was attributed to a better extraction of polyphenols due to the presence of ethanol, rather than to a real increase in their content. Moreover, this method allowed the detection of three unknown antioxidant compounds, which accounted for 64 ± 4% of the total antioxidant activity of beer and were individually more efficient than caffeic acid and epicatechin.