The polyphenol composition and antioxidant activity of seven Australian-grown barley varieties were characterized in this study. UHPLC with an online ABTS system was used to identify individual polyphenols while simultaneously measuring their antioxidant activity. The Q-TOF LC/MS system was utilized to identify the phenolic compounds that demonstrated substantial antioxidant activity. The variety, Hindmarsh, showed the highest total phenolic content and antioxidant activity. There was no significant difference observed amongst the other varieties in their total phenolic content, however, they did have significant variation in proanthocyanidin content and antioxidant activity (p < 0.05). Prodelphinidin B3 was the most abundant polyphenol with the highest antioxidant activity amongst all the barley varieties tested. Other polyphenols identified with antioxidant activity included procyanidin, glycosides of catechin and flavan-3-ols. Polyphenol characterization of Australian grown barley varieties demonstrated that they have significant antioxidant activity, hence, promoting the value of whole grain barley as a potential functional food ingredient.