Although the drugs that achieve their effect by covalently binding the target molecules have their advantages, the risk of toxicity caused by nonspecific binding poses complications in their development. Some of those drugs are Michael acceptors. They are electrophiles that covalently bind cysteine residues of target proteins. In addition to toxicity caused by the modification of bound protein's properties, nonspecific covalent binding of Michael acceptors to protein and peptide thiol groups leads to glutathione depletion. Glutathione depletion damages the function of antioxidant defense mechanisms and causes the state of oxidative stress. In this paper, the effect of eleven potential cytostatics on the concentration of thiol groups in human serum was tested. The tested compounds are itaconic acid derivatives and Michael acceptors. Antimalarics that are structural components of some derivatives were also tested. The tested compounds were added to the samples of human serum. Thiol group concentration was determined after a one-hour incubation. Each compound was tested with an incubation at 37 °C and with an incubation at room temperature. Thiol group concentration was determined using the Ellman reagent method. Substantial decrease in thiol group concentration indicates nonspecific binding of the tested compound. Out of eleven tested itaconic acid derivatives, only two compounds, V9 and MI9, didn’t cause a decrease in thiol group concentration greater than 30% at either temperature. None of the tested antimalarics caused a decrease in thiol group concentration greater than 10% at either temperature. Additional testing is required for further characterization of in vivo nonspecific covalent binding burden of the tested itaconic acid derivatives.