The blood-feeding invertebrates are a rich biological source of drugs and lead compounds to treat cardiovascular diseases, as they have evolved highly efficient mechanisms to feed on their hosts by blocking blood coagulation. In this work, we have focused our attention on the leech Hirudo medicinalis. We performed, by ‘Intensity-Fading’ (IF) MALDI-TOF mass spectrometry, a comprehensive detection and functional analysis of pre-existent peptides and small proteins with capability of binding to trypsin-like proteases related with blood coagulation. Combining ‘Intensity Fading MS’ and off-line liquid chromatography (LC) pre-fractionation allowed us to detect more than 75 molecules present in the leech extract that interacts specifically with a trypsin-like protease over a sample profile of nearly 2,000 different peptides/proteins in the 2-20 KDa range. Moreover, we have resolved 232 individual components from the complex mixture, 13 of which have high sequence homology with previously described serine proteases inhibitors. Our findings indicate that such extracts are much more complex than expected. Additionally, ‘Intensity Fading MS’, when complemented with LC separation strategies, seems to be a useful tool to investigate complex biological samples, establishing a new bridge between profiling, functional peptidomics and subsequent drug discovery.