In this study we investigated the proteolysis of human plasma proteins by HF3, an hemorrhagic metalloproteinase from B. jararaca venom. For this purpose, approaches for the depletion of the most abundant proteins and for the enrichment of low abundant proteins of human plasma were used to minimize the dynamic range of protein concentration, in order to assess the proteolytic activity of HF3 on a wide spectrum of proteins, and to detect the degradation products by mass spectrometry by untargeted peptidomics.