In this study, we established the pre-treatment conditions that could increase the γ-aminobutyric acid (GABA) content during barley germination. In the process, three different barley samples were prepared, which differed in the pre-treatedment processes. The specimens were stored at 50 for 1 h after being kept in water at room temperature for 4 h (HKW sample), kept in sufficient water for 4 h (KW sample), or left untreated (CO sample). After the pre-treatment, the barely samples were germinated for 35 h. A sample was taken from each batch in 5-h intervals, extracted with water, and physicochemical characteristics and radical scavenging activity were measured. As a result, we found that the contents of phenolic compounds (18.02-30.63 mg/100 g) and flavonoids (1.87-4.63 mg/100 g) were higher in HKW, showing similar trends. Also the GABA contents in the HKW and KW samples were higher than that in the CO sample. Furthermore, the radical scavenging activities of DPPH and ABTS were the highest in the HKW sample, having values in the ranges of 58.49-77.40% and 54.57-88.10%, respectively. All in all, we found that in order to increase the antioxidative activity and GABA content of the barley samples, it would be suitable to pre-treatment the specimens after the post-immersion heat treatment. In addition, pre-treating the KW samples is appropriate only after immersion time. Lastly, the optimum germination time of the batches was found to be 20-25 h.