Barley’s nutritional value as a health food is increasing due to its excellent nutritional functionality. In this study, the levels of β-glucan, total polyphenols, and total flavonoids were analyzed in the ethanol extracts of different barley cultivars (Hinchalssal, Heuksoojeongchal, Betaone, Ganghochung, and Saechalssal). Also, the free radical scavenging abilities of 2,2-diphenyl-1-picrylhydrazil (DPPH) and 2,2’-azino-bis-3-ethylbenzo-thiaxoline-6-sulfonic acid (ABTS) were measured to determine their antioxidant activity. The results confirmed that Betaone extract contained highly activefunctional components and exhibitedantioxidant activity. Next, we evaluated the hepatoprotective and inhibitory effects of reactive oxygen species (ROS) generated by barley ethanol extracts after inducing oxidative stress with tert-butyl hydroperoxide (tBHP) in HepG2 cells. Hinchalssal and Saechalssal extracts showed the most significant cytoprotective effect and also reduced ROS production significantly. These results suggest that Hinchalssal, Saechalssal, and Betaone represent potential natural antioxidant and hepatoprotective agents.