Present study was aimed at the evaluation of the endothelial cytotoxicity of the plasminogen activator inhibitor type-1 mutated protein characterized by the very long half-life time (VLHL PAI-1) and therefore prolonged anti-proteinase activity. Viability of the human umbilical vein endothelial cells (HUVECs) cultured for 72 hours with the increasing concentrations (1, 10, 100 µg/ml) of VLHL PAI-1 was not affected as demonstrated by the neutral red test and direct cell counting. On the contrary, the classical MTT test proved to be unreliable for this experimental setting most possibly due to the cell specific VLHL PAI-1 – HUVECs interaction. This suggestion was further confirmed by the similar results of the viability assessment by the MTT test and direct cell count in the A549 and NCI-H1299 lung cancer cell lines cultures with VLHL PAI-1 mutated protein.