The overproduction of reactive oxygen species (ROS) causes oxidative stress, such as Hydrogen peroxide (H 2 O 2 ). Acute oxidative stress is one of the main reasons for cell death. In this study, the antioxidant properties of vanillic acid- a polyphenolic compound was evaluated. Therefore, this study aims to check the effectiveness of vanillic acid in H 2 O 2 -induced oxidative stress in D. Mel-2 cell line. The efficacy was determined by biochemical tests to check the ROS production. The cytotoxicity of H 2 O 2 and vanillic acid was checked by MTT assay. The DNA fragmentation was visualized by gel electrophoresis. Protein biomarkers of oxidative stress were analyzed by western blotting. The results depict a promising antioxidant effect of vanillic acid. The IC 50 value of vanillic acid and H 2 O 2 was found 250 μg/ml and 125 μg/ml, respectively. The catalase activity, SOF, GPx, and PC was seen less in H 2 O 2 treated group compared with the control and vanillic acid treated group. However, the TBRAS activity was hight in H 2 O 2 treated group. The effect of H 2 O 2 on DNA fragmentation was high as compared with vanillic acid-treated cells. The protein expression of Hsp70, IL-6 and iNOS was seen significant in a vanillic acid-treated group as compared with H 2 O 2 treated group. These results reinforce that at low concentration, vanillic acid could be used as an antioxidant agent in the food and pharmaceutical industries.