Lionfish (Pterois volitans) was classified as an invasive predator native to the Indo-Pacific region that has no natural predators. Lionfish grow fast, making these fish prey on much smaller fish in which causing damage to the marine ecosystem. This research determines the antioxidant potential of lionfish spines venom by isolating the protein from its extract. In previous research, it was specified that lionfish poison extract have had the potential as an antioxidant, however, it was still feeble. In order to find out the potential further, the additional isolation step is heating. Crude venom (CV) was extracted using sonication and isolated by heating at 90°C, 75°C, and 60°C followed by fractionated using Ammonium Sulfate (AS) with 0-20, 20-40, 40-60, and 60-80% saturation. The concentrated protein was then analyzed by the Lowry test and the protein content was identified using SDS-PAGE, followed by toxicity test using BSLT method (Brine Shrimp Lethality Test). The antioxidant activity was carried out using the DPPH method in the final step. The optimum condition for isolating proteins which have the potential to have antioxidant activity observed in specimen with a heating temperature of 75 and saturation of ammonium sulfate 40-60% with an IC50 value of 1312 ppm. The protein composition at the protein isolation temperature for optimum antioxidant is protein 7.9; 46.2; and 52.7 kD.