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Bothrops asper envenoming is a public health problem in tropical regions of Latin America. Bothrops asper has spread until Gorgona Island in the Pacific Colombian Ocean, but its biochemical venom characterization is poorly known. Thus, to increase knowledge on Bothrops species venoms, we developed for the first time the proteomic analysis using a shotgun approach and performed functional evaluations relevant to its toxicity and compared with two Colombian Southwest ecoregions from the Pacific and Western sides. Besides, we evaluated two antivenoms produced in Colombia (INS and PROBIOL) against three B. asper venom ecoregions through the ELISA approach and first-generation antivenom against B. asper from Gorgona Island. The protein components of B. asper from Gorgona Island were assigned to nine known protein families, sharing a conserved compositional pattern with B. asper from the pacific ecoregion. The RP-HPLC and in vitro activity suggest a phenotypic congruence in the expression of PLA