Introduction: Long term consumption of antioxidant supplements has been shown to be related to adverse effects such as increased risk of death from synthetic antioxidant compounds used. Of all Indonesia’s marine wealth, Scorpaenopsis diabolus is still rarely studied regarding its bioactive components in its venom. To evaluate the purification process and the potential of S. diabolus toxin as natural antioxidant. Materials and Methods: Toxins were extracted by batch method using phosphate buffered-saline and purified using FPLC system with anion exchanger column. In order to identify the concentration and molecular weight of protein, Lowry analysis and SDS-PAGE test were carried out. The protein was then assayed for toxic activity using the Brine Shrimp Lethality Test and assayed for antioxidant activity using DPPH method (2,2-diphenyl-1-picrylhydrazil). Results: Ten purified protein fractions were obtained from S. diabolus toxin with the highest concentration in 101.1 kDa fraction from 0% salt elution. The highest toxic activity was found in 25 – 225 kDa fraction with LC50 = 27.68 μg/mL, while the antioxidant activity varied from weak to strong with IC50 values ranging from 64 – 179 μg/mL, but with a negative inhibition percentage, except for protein with an estimated molecular weight of ~10 kDa at 50 ppm. Conclusion: Based on its IC50 value, Scorpaenopsis diabolus venom has the potential to be developed as antioxidants. However, research regarding the antioxidant activity of other protein fractions still need to be done, and further study using more advanced antioxidant testing method is required, as most of the inhibitory activity are negative. © 2025 Universiti Putra Malaysia Press. All rights reserved.