Ritalin, [(+)-threo]methylphenidate hydrochloride, is a chiral drug substance with two chiral centers. The drug substance may contain three pairs of enantiomers, [(+)-threo], [(-)-threo], [(+)-erythro] and [(-)-erythro] isomers, and its degradation products, threo-ritalinic acid racemate. Determination of the optical purity of ritalin drug substance and the amount of its by-product isomers is a critical step in the single-isomer drug development. In order to efficiently recognize the three pairs of enantiomers by one method, capillary electrophoresis (CE) was employed for the separation. The three pairs of enantiomers in CE showed different enantioselectivities with eight different types of CDs. Only 2,6-di-o-methyl-beta -cyclodextrin (DM-beta -CD) and carboxymethyl-beta -cyclodextrin (CM-beta -CD) showed enantioselectivity to all these pairs of enantiomers. With respect to separation resolution and efficiency, DM-beta -CD was chosen as the chiral selector. For optimization of the separation conditions, the concentration of DM-beta -CD, pH of the buffer solution, and temperature of the capillary were further studied.