Abstract A capillary electrophoresis method for determining the enantiomeric purity of moxifloxacin hydrochloride in drug substance and ophthalmic/otic drug products was developed and validated. Because moxifloxacin hydrochloride has two chiral centers, the existence of four different isomers is possible. The method was capable of separating moxifloxacin hydrochloride, which is the S, S-isomer, from its potential chiral degradation products, which are the R, R-enantiomer, the R, S-diastereomer, and the S, R-diastereomer. The separation was carried out at 20 °C in a 50 μm × 40 cm fused-silica capillary with an applied voltage of −13 kV using a 12.5 mM TEA phosphate buffer (pH 2.5) containing 5% highly-sulfated gamma-cyclodextrin (HS-γ-CD) and 6% acetonitrile. The detection wavelength was 295 nm. The method was validated with respect to its specificity, linearity, range, accuracy, and precision in the expected range of occurrence for the isomeric impurities (0.05–5%). The method was used to investigate whether racemization was a potential degradation pathway for the drug substance, both in the solid state and in solution.