Snake venom serine proteinases (SVSPs) belong to the trypsin/chymotrypsin subfamily of enzymes, share high sequence identities (50-70%) and are highly specific in relation to the macromolecular substrates with which they interact. These enzymes are glycosylated to varying degrees and interfere in the control, regulation and maintenance of the hemostatic system by interacting with the enzymes of the coagulation cascade and the fibrinolytic feedback system. Procoagulant SVSPs have received much attention due to their potential clinical applications since they are capable of converting fibrinogen to fibrin by the specific cleavage of fibrinopeptide A (Aa 1-16) from the N-terminal portion of the Aa-chain. These SVSPs are also referred to as thrombin-like enzymes because they form a non-cross linked “soft clot” that is rapidly eliminated from the circulatory system by the fibrinolytic mechanism resulting in a defibrinogenerating effect. On the other hand, PA-BJ isolated from the venom of Bothrops jararaca possesses platelet aggregating activity. With the aim of understanding the specificities of SVSPs and the role of the carbohydrate moieties, we have recently solved the crystal structure of the protein C activator from the venom of Agkistrodon contortrix contortrix and now extend our study to include the development of experimental techniques for the establishment of large-scale purification of SVSPs from the venoms of Bothrops jararaca, Crotalus durissus terrificus and Crotalus durissus collilineatus that exhibit fibrinogen-clotting and platelet aggregating activities. The purification procedures involved a combination of gel filtration, ion-exchange and affinity chromatography (benzamidine-sepharose 4 fast flow). After each step, the specific activity of the enzymes was monitored and the purity of the samples was confirmed by silver stained SDS-PAGE gels and mass spectrometry. Crystals suitable for the determination of the crystal structures have been obtained and diffraction data has been collected.