ABSTRACT Snake venoms are important sources of substances with a variety of pharmacological activities. Among the different proteins present in these venoms, snake venom serine proteinases (SVSPs) have important effects on the hemostatic system that influence the hemodynamic properties of blood. Bothrops genus snakes presented their venom richly composed of SVSPs thrombin-like, and the isolation of these enzymes is of great interest. In 1994, the Center for the Study of Venoms and Venomous Animals (CEVAP) - UNESP standardized the fibrin sealant derived from snake venom, replacing the bovine thrombin by gyroxin thrombin-like enzyme from Crotalus durissus terrificus (Rattlesnake) and human plasma fibrinogen by buffaloes cryoprecipitate. Despite chromatographic techniques for the purification of gyroxin be well grounded in the literature, that income is considered low. Thus, in addition to gyroxin, other thrombin-like enzymes could be employed in the composition of the new fibrin sealant after being standardized to the purifying and chromatographic performance and widely evaluated for biological activities. Therefore, it is extremely important that in our lab is deployed, standardized and validated a method for the chromatographic purification of other thrombin-like enzymes such as found in Bothrops snake venoms. Thus a two-step chromatographic procedure was developed to routinely purify serine proteinases from Bothrops alternatus and B. moojeni snakes venoms to provide new enzymes for improving the CEVAP’s heterologous fibrin sealant.