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Phospholipase A₂ (PLA₂) toxins are one of the main toxin families found in snake venom. PLA₂ toxins are associated with various detrimental effects, including neurotoxicity, myotoxicity, hemostatic disturbances, nephrotoxicity, edema, and inflammation. Although <i>Naja sumatrana</i> venom contains substantial quantities of PLA₂ components_, there is limited information on the function and activities of PLA₂ toxins from the venom. In this study, a secretory PLA₂ from the venom of Malaysian <i>N. sumatrana</i>, subsequently named A2-EPTX-Nsm1a, was isolated, purified, and characterized. A2-EPTX-Nsm1a was purified using a mass spectrometry-guided approach and multiple chromatography steps. Based on LC-MSMS, A2-EPTX-Nsm1a was found to show high sequence similarity with PLA₂ from venoms of other <i>Naja</i> species. The PLA₂ activity of A2-EPTX-Nsm1 was inhibited by 4-BPB and EDTA. A2-EPTX-Nsm1a was significantly less cytotoxic in a neuroblastoma cell line (SH-SY5Y) compared to crude venom and did not show a concentration-dependent cytotoxic activity. To our knowledge, this is the first study that characterizes and investigates the cytotoxicity of an Asp49 PLA₂ isolated from Malaysian <i>N. sumatrana</i> venom in a human neuroblastoma cell line.