Venoms from Bothrops genus snakes are recognized by their action on hemostatic system. The identification of proteins that interact with hemostatic system molecules is a strategy of great importance for the discovery of new compounds with potential therapeutic use. The aim of this work is to identify proteins from Bothrops jararaca venom interacting with purified prothrombin and thrombin, from human plasma, through a proteomic approach. Prothrombin and thrombin were purified of the human plasma following a previously established protocol. After the purification, each molecule was incorporate into a 5 ml CNBr - Sepharose 4 B column and Bothrops jararaca venom (20 mg and 100 mg) was applied respectively in prothrombin and thrombin affinity columns. The fractions retained in these columns were eluted, dialyzed, concentrated and applied (250 mg) into two-dimensional (2D) gel (pH 3-10) for isoelectrofoccusing and 12,5 % for SDS-PAGE) stained with Colloidal Coomassie. Approximately 15-20 spots were detected in each gel, excised and hydrolyzed with trypsin and then analyzed by MS and MS-MS experiments, with MALDI-TOF (VoyagerDE PRO-Applied Biosystems) and MALDI TOF-TOF (ABI 4700 Proteomics Analyser Applied Biosystems). The NCBI database was used for identification of venom proteins that interact with prothrombin and thrombin. Different classes of proteins were identified such as metalloproteases (jararagin), serino-proteinase (platelet-aggregating proteinase PA-BJ) and lectins. The formation of the complex between the Bj galactose C-type lectin and prothrombin was confirmed by native gel electrophoresis. We concluded that the combination of affinity chromatography with the proteomic technique is efficient in identifying venom proteins that interact with blood components leading to the discovery of new protein x protein interactions. Also this results can guide to a new understanding of the envenomation process. Financial support: FAPERJ, CNPq.