Cottonseeds are fed to high-producing dairy cows as a source of fat and highly-digestible fiber. Previously we reported the isolation and structure characterization of different phenolic compounds, quercetin, kaemferol and benzoic acid derivatives (1-9) from whole cottonseed by-product. In this paper the antioxidant activity of all isolated compounds was determined by measuring free radical scavenging effects using the Trolox equivalent antioxidant capacity assay and the coupled oxidation of β-carotene and linoleic acid (autoxidation assay). The range of phenolic compounds present in WCS (1.90 g/kg), assayed by the Folin-Ciocalteu method, and their antioxidant properties provides a valuable database for possible nutritional studies on the impact of diet on animal health and productivity.