The aim of our study was to optimize quantitative proteomic analysis of fibrin clots prepared ex vivo from citrated plasma of the peripheral blood drawn from patients with prior venous thromboembolism. We used a multiple enzyme digestion filter aided sample preparation (MED FASP) method combined with LC-MS/MS analysis performed on a Proxeon Easy-nLC System coupled to Q Exactive HF mass spectrometer. We also evaluated the impact of peptide fractionation with pipet-tip strong anion exchange (SAX) method on the obtained results. Our proteomic approach revealed >500 proteins repeatedly identified in the plasma fibrin clots from patients with venous thromboembolism. The multienzyme digestion (MED) FASP method using three different enzymes: LysC, trypsin and chymotrypsin increased the number of identified peptides and proteins and their sequence coverage as compared to a single and double step digestion. Peptide fractionation with SAX protocol slightly increased the depth of proteomic analyses, but also extended the time needed for sample analysis with LC-MS/MS.