Alkoxyacetic acids (AAAs) are known urinary metabolites of the corresponding ethylene glycol monoalkyl ethers with a wide range of industrial and domestic applications. Hemolysis is the principal toxic effect of AAAs in humans and animals. The mechanism of red-cell damage is not known. It is suggested that some disturbances in ion balance, mainly related to calcium are one of the reasons of hemolysis. No comparative studies in the available literature on the chelating properties of numerous AAAs in respect to calcium were found. Therefore, a comparison was made between chelating effects of five AAAs on calcium and magnesium in vitro. It was demonstrated that calcium was bound at lower AAAs concentrations than magnesium. The chelating effect of AAAs expressed by EC50 values was positively correlated with both pKa values and Log P values of the examined acids. The obtained data indicate that the acidity and hydrophilic properties are responsible for the chelating effect of AAAs on calcium and magnesium in vitro. These data do not provide an explanation for differences in the hemolytic activity of the examined compounds.