The ever-increasing research efforts to develop new antithrombotic therapies have led to the reassessment of the role of alpha-2-plasmin inhibitor (α2-PI) in pathological conditions. In particular, experimental stroke studies have suggested correlation between increased free α2-PI level and mortality. However there are only a small number of well-characterized and specific assays available for the measurements of free α2-PI. In plasma α2-PI undergoes both N- and/or C-terminal cleavages resulting four isoforms with modified susceptibility to FXIII catalyzed cross-linking to fibrin and/or loss of plasmin(ogen) binding. Present paper describes a new sandwich ELISA method for the determination of free total α2-PI in plasma and other body fluids. A newly generated biotinylated monoclonal antibody recognizes and captures all the four N- and/or C-terminally modified isoforms of α2-PI while HRPO-labeled polyclonal anti-α2-PI antibody detects the captured antigen. Performing the 2-step assay in streptavidin-coated microplate can be completed within three hours. The assay is well reproducible, total (within laboratory) imprecision in the normal, pathological and very low ranges were 7.4%, 9.1% and < 19%, respectively. When examining the plasma samples of 197 healthy volunteers, 100 acute ischemic stroke patients and 102 patients with venous thrombosis, strong correlation was observed between total α2-PI antigen levels and α2-PI activity for each group. Using the assay a reference interval of 45-86 mg/L was established for total α2-PI mass concentration in the plasma. α2-PI levels were also measured in cerebrospinal fluid samples of 47 individuals the median value and range was 132 (36-379) μg/L. In conclusion, our ELISA enables accurate and fast measurement of total free α2-PI in human body fluids.