A chiral capillary electrophoresis assay for the simultaneous determination of the optical purity and of related substances of calcium levofolinate has been developed and. validated. Using 2,6-dimethyl-beta-cyclodextrin as chiral selector at a concentration of 20 mg/mL, the method was optimized using a full factorial design with four factors including pH and concentration of the background electrolyte, column temperature and separation voltage. Optimized conditions were a 40 mm sodium tetraborate buffer, pH 9.9, a capillary temperature of 16degreesC, and an applied voltage of 21 kV. Methotrexate was used as internal standard to compensate for injection errors and fluctuations of the migration times. A multiple linear regression model was also used to test the robustness of the method. Validation was performed with respect to specificity, linearity, range, limit of quantification and detection, precision, and accuracy. The assay allowed the detection and determination of related substances including the diastereomeric (6R,2'S)-impurity of levofolinic acid at the 0.1% level, the identification threshold of impurities for orally administered drugs for human use defined by the International Conference on Harmonization guidelines as well as the European Pharmacopoeia.