Lionfish Pterois volitans is a tropical Indo-Pacific native species and invaded on marine ecosystem due to its rapid growth and predatory properties that were highly affecting fish and coral populations. Recent studies show the benefits of natural antioxidants, especially as the prevention of cancer and cardiovascular disease. Crude Venom (CV) was extracted using sonication and precipitated using ethanol and Ammonium Sulfate (AS) with 20, 40, 60, and 80% saturation. The protein concentration of each sample detected using Lowry's method, and SDS-PAGE was done to find out the Molecular Weight (MW) of protein samples. Toxicity analysis to determine the Lethal Concentration (LC50) value was done using Brine Shrimp Lethality Test (BSLT) and antioxidant activity tested using 2,2-diphenyl-1-picrylhydrazyl (DPPH) method to determined Inhibitory Concentration (IC50) value. AS 80% had the higher value of protein concentration 361.76 μg/mL. SDS-PAGE analysis showed that AS 80% had a protein band at 7.9, 46.2 and 52.7 kD which supposed to be the protein that had antioxidant activity. The test showed the LC50 of AS 80% 101.93 μg/mL. Antioxidant analysis showed AS 80% had the higher value of inhibition 57.08 % with IC50 1.56 mg/mL. This research showed that AS 80% has the highest potential as an antioxidant compound due to its ability to inhibit free radicals.