Drug development is a complex process. It begins with the screening of molecules by computer modeling, by which the molecules with potential therapeutic effects are isolated. It is followed by in vitro experiments which are usually carried out on specific tissues or cells. These experiments are of great importance because if the tested molecules do not show the desired activity, further development of the molecules is halted and in vivo and clinical trials are not performed. The cytotoxicity test, which is crucial in the development of cytostatics, belongs to the group of in vitro studies. Cytostatics act by killing tumor cells. They are non-selective, i.e. toxic to healthy cells and cause severe side effects. Therefore, by reducing the dose of cytostatics, side effects are also reduced. Due to the lack of effectiveness of existing tumor therapies, scientists are looking for new, more effective drugs. The topic of this thesis was to test the cytotoxicity of itaconic acid derivatives as potential cytostatics. The study was performed on hepatocellular carcinoma HepG2 cells. Cells were treated with a solution of synthetic compounds in various concentrations (100 μM, 50 μM, 25 μM, 12.5 μM, 6 μM, 3 μM, 1.5 μM, 0.8 μM). Cell viability was determined by using an MTT assay in which metabolically active cells convert MTT to a purple-colored compound formazan, whereas the dead ones lack this ability. The amount of produced formazan, which is proportional to the number of living cells, is determined spectrophotometrically. To simplify cytotoxicity comparisons, the results are presented in the form of IC50 values. All test compounds were cytotoxic for hepatocellular carcinoma cells HepG2. The most effective compound, toxic even at low concentrations, is V4. This finding is important because if the compound passes other stages of the development, it could be administered at low doses.