Effect of plant defense activator on Polygonum tinctorium was evaluated to increase indigoid content in P. tinctorium. Three plant defense activators, 2,1,3-benzothiadiazole (BTH), 2,6-dichloroisonicotinic acid (INA), and acibenzolar-S methyl (ASM) were treated to two cultivars (Naju Local and Naram Blue) under the field condition at two different concentration regimens of 0.3 and 3 mM and harvested 7 days after treatment. The treatments increased indigo accumulation by 40 to 109% with statistical significance. Considering the treatment concentrations, BTH and INA at 0.3 mM were judged to be optimal with 2-fold increase in indigoid concentration. No statistical differences were found in the cultivars and maturity of leaves in the indigoid accumulation caused by plant activator treatment. This result is directly applicable to the P. tinctorium cultivation with high economic significance.