Background: A Cobra’s venom is a complex mixture of pharmacologically active proteins. They play an essential role in incapacitating, immobilizing, and digesting of prey. Many Cobra venoms contain anticoagulant toxins which prevent blood clotting and importantly help neurotoxins spread and reach their targets. Objective: This study aims at the application of nanofractionation analytics for unravelling the Black Spitting Cobra (Naja nigricollis) venom toxin responsible for venom-induced coagulopathic toxicities after envenoming. Materials and Methods: An integrated analytical approach was used in which venoms were analyzed by liquid chromatography coupled with mass spectrometry (MS) followed by nanofractionation on a 384-well plate for bioassaying coagulopathic properties of eluting venom constituents. After centrifuging the fraction on the 384-well plates, a spectrophotometric bioassay kinetically measured coagulation for 1.5 h after rapid automated pipetting of 20 μL of CaCl2 (20 mM) followed by 20 μL of citrated bovine plasma. Calcium ions titrate away the citrate in the plasma and thereby initiate coagulation. Data processing involved plotting the coagulation potency measured for each well against the fraction time to create bioactivity chromatograms. Proteomics analysis was also performed to the fractionated venom using in-solution tryptic digestion followed by MS/MS measurements. MASCOT database was used for comparing the MS/MS spectrum to identify the peptides/proteins. Results: In the wells showing anticoagulation activity, MS analysis revealed that Naja nigricollis venom was found to consist mostly of acidic phospholipase A2 (PLA2) proteins and many three-finger toxins were identified. It was found that the venom also contains metalloproteinases as minor components. An inhibition assay was performed against PLA2s using varespladib as a specific inhibitor. Conclusion: We conclude that PLA₂s, three-finger toxins, and metalloproteinases are the most likely candidates responsible for the observed anticoagulant properties of Naja nigricollis venom.