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Abstract: The objective of this study was to determine the phytochemical profiles and antioxidant activities of three highland barley varieties. The free, bound, and total phenolic content of highland barley, respectively ranged from 44.52 to 178.37 mg gallic acid equivalents (GAE)/100 g dry weight (D. W.), 121.69–302.55 mg GAE/100 g D. W., and 166.21–480.92 mg GAE/100 g D. W. Besides, the free, bound, and total flavonoid content, respectively, ranged from 15.71 to 35.18 mg rutin equivalents (RE)/100 g D. W., 39.37–86.93 mg RE/100 g D. W., and 59.13–102.64 mg RE/100 g D. W. The 2,2‐diphenyl‐1‐picrylhydrazyl radical scavenging abilities, oxygen radical absorbance capacities, ferric reducing antioxidant power values, and cellular antioxidant activities of the bound extractions of highland barley were higher than those of the free extractions. However, the free polyphenol extracts had stronger inhibitory effects on HepG2 cell proliferation than the bound extracts. Practical applications: Highland barley is a principal food crop that is widely cultivated in the west and southwest regions of China. Highland barley is usually rich in phenolic compounds, however, the phenolic compounds content and antioxidant activities of highland barley were underestimated because the bound phytochemical fraction was not included in the analysis. In this study, three highland barleys from different region of China were used to analysis the free and bound total phenolics and total flavonoids, as well as the antioxidant activities. [ABSTRACT FROM AUTHOR]