This research aimed to study the synthesis of hydroxyl radicals in form of plasma-activated water by gliding arc plasma. Initially, plasma experiments were conducted to confirm the occurrence of hydroxyl radical by using optical emission spectrophotometer, and then compared the use of distilled water and microbubble as raw material for the synthesis of hydroxyl radicals. Then, the existence of hydroxyl radicals in the form of hydrogen peroxide was studied. Finally, the factors that affect the synthesis of hydroxyl radical from are investigated by using fractional factorial experiments. Plasma measurements show that the peak at 310 nm occurs, meaning that the gliding arc plasma can synthesize hydroxyl radicals. It was found that the use of microbubble as a raw material could better synthesize hydroxyl radicals than distilled water. In addition, the existence of hydroxyl radical in the form of hydrogen peroxide is likely to decrease with time. Finally, the factors that affect the synthesis of hydroxyl radicals are amount of sodium bicarbonate, gap between electrodes, argon flow rate, and oxygenation ratio.