Lionfish (Pterois volitans) are a fish that live in the Indo-Pacific region. Today, Lionfish have an increasing population and invaded up to the Atlantic Ocean. The invasion of Lionfish was not accompanied by the presence of predators, thus causing damage to the ecosystem and food chain. One of the control efforts that possible be done is the utilization of venomous spines of Lionfish that contains Phospholipase A2 known to have antiretroviral activity. So it has the potential to be used as an alternative treatment of HIV/AIDS infection. This research aims to increase the purity and specific activity of Phospholipase A2 from venomous spines of Lionfish in the Java Sea, Indonesia. Phospholipase A2 isolated from Lionfish venom from the waters of the Java Sea with Salting-out method using Ammonium Sulfate on 20% saturation level and the addition of 0.5 mL of Caprylic Acid by heating 60 °C for 20 minutes has the highest enzymes activity and purity. The specific activities reaching 6.48 units/μg, safe tested on human cells with 28.36 ppm inhibition measured with LC50 and also can inhibit infected cell growth with 98.13% inhibition degree.