Glycolysis is for some cells, such as erythrocytes, neutrophil granulocytes and many cancer cells, the only or most important source of energy (ATP) production. Based on previous studies we developed an isotachophoretic (ITP) method which allows, in principle, the simultaneous determination of all metabolites of glycolysis. Since glucose metabolites are small anions, mobility of some of them may overlap in isotachophoresis and, therefore, partial mixed zones are generated. By variation of the leading/terminating system, however, it is possible to separate the compounds of interest. In this communication, we describe a method for analysis of glucose metabolites in erythrocytes from healthy donors during storage in blood bags, and from patients with thalassemia, with special respect to intracellular 2,3 bisphosphoglycerate, lactate and ATP/ADP. The well known characteristic changes of glycolysis in erythrocytes during blood storage and in erythrocytes from thalassemia patients, which are often analysed by separate enzymatic assays, could be confirmed with this isotachophoretic procedure. The method is currently adapted for analysis of glycolysis in neutrophil granulocytes and cancer cells which requires some modifications of sample preparation and performance of the isotachophoretic analysis.