The single enantiomer drug, alogliptin (Alo, Nesina (R)) is a novel, orally available and selective dipeptidyl peptidase-4 inhibitor used for the treatment of type II diabetes. Following its pKa determination by CE-pH titration, a validated chiral CE method has been developed to separate Alo enantiomers. Preliminary screening of the native CDs and their ten derivatives revealed that sulfopropylated--CD, sulfopropylated--CD and sulfopropylated--CD, sulfobutyl-ether--CD (SBE--CD) and sulfobutyl-ether--CD enabled enantioresolution. Furthermore, cavity size dependent enantiomer migration order reversal was observed between - and -CD derivatives. To improve enantioseparation, buffer composition and pH, CD concentration, applied voltage, temperature, and injection parameters were optimized for the Alo/ SBE--CD system, yielding a resolution of 8.34. RSD percentage of the resolution value, migration times, and corrected peak areas were below 3 and 5% during testing repeatability and intermediate precision. LOD and LOQ values were found to be 2 and 6 g/mL, respectively, for each enantiomer. Calibration lines ranging from 6 to 250 g/mL were constructed with r(2) > 0.9997. Robustness could be successfully verified by using the Plackett-Burman statistical experimental design. The optimized system containing 5mM SBE--CD in a 25mM acetate buffer at pH 4.75 was found promising to detect 0.1% distomer in the presence of the API.