This study characterizes the substrate specificity and membrane interactions of heterodimeric phospholipase A₂ (HDP-2) from Vipera nikolskii venom through combined biochemical and computational approaches. Using model membrane systems comprising pure phospholipids (phosphatidylcholine, cardiolipin, phosphatidylserine, and sphingomyelin) and physiologically relevant mixed membranes, we demonstrate that HDP-2 exhibits distinct hydrolytic preferences. The enzyme shows greater activity toward pure cardiolipin than phosphatidylserine, while in mixed membranes this preference reverses, with enhanced hydrolysis observed in phosphatidylcholine- phosphatidylserine systems compared to phosphatidylcholine-cardiolipin membranes. Notably, sphingomyelin-rich membranes resist hydrolysis unless combined with phosphatidylcholine. AutoDock simulations reveal the structural basis for these observations, showing productive binding of phosphatidylserine, phosphatidylcholine, and cardiolipin to HDP-2’s active site, with optimal positioning of their sn-2 ester bonds for catalysis. In contrast, sphingomyelin binds peripherally, explaining its resistance to hydrolysis. These findings align with the enzyme’s physiological targets, suggesting preferential attack on cardiolipin-rich mitochondria of cardiomyocytes and phosphatidylserine-exposing membranes in autophagy, while sparing sphingomyelin-rich neural membranes.Compared to its homolog HDP-1 (known to affect cardiac contractility), HDP-2 appears optimized for disrupting cardiomyocyte membranes during envenomation. The differential hydrolysis of phosphatidylserine versus sphingomyelin suggests dual pharmacological roles: phosphatidylserine may amplify apoptotic signaling through enhanced phospholipase A₂ activity, while sphingomyelin’s resistance to hydrolysis may provide inherent neuroprotection against excessive membrane degradation. These results highlight how membrane lipid composition and packing density critically regulate HDP-2’s neurotoxic potential, with important implications for understanding viper envenomation pathophysiology and developing targeted therapies.