Lionfish (Pterois volitans) is a native species of the Indo-Pacific Ocean. This species is known as destructive invasive species that invade in many areas. The invasion of Lionfish gives negative impacts on the ecology and economic condition of the invaded regions. Therefore, to reduce the number of Lionfish in the ocean and to attract people to exploit Lionfish, the study about Lionfish's benefit is necessary. The recent study revealed that venom extract from Lionfish spines has a potential cytolytic effect that widely uses for antitumor studies. In this study, to obtain the optimum concentration of the solvent that used for protein isolation and to observe the antitumor properties of Lionfish further, we isolated the protein of Lionfish venom extract with ammonium sulfate and tested it on HeLa cell as the model of tumor cells. The Lowry and BSLT test showed that the highest protein concentration and activity obtained when the highest level of ammonium sulfate isolated crude venom. The highest percentage of inhibition in HeLa cells reach 17-19% at the highest dose of Lionfish protein isolate. Indeed, the inhibition effect on HeLa cells can be enhanced because SDS-PAGE results showed that there are several proteins with different effects on tumor cells contained in the protein isolate of Lionfish venom. Thus, eliminating these proteins might increase the inhibition effects of Lionfish venom protein in tumor cells. These results suggest that Lionfish has an antitumor effect, but need further purification step and more evaluation of the protein in Lionfish venom extract that can enhance the antitumor effect.