The single enantiomer drug, alogliptin (Nesina®) is a novel, orally available and selective DPP-4 inhibitor used for the treatment of type II diabetes. Following its pKa determination by CE-pH titration, a validated chiral capillary electrophoresis (CE) method has been developed to separate alogliptin enantiomers. Preliminary screening of the native cyclodextrins (CDs) and their 10 derivatives revealed that γ-, sulfopropylated-β- and -γ-, sulfobutyl-ether-β- and -γ-CD enabled enantioresolution. Furthermore, cavity size dependent enantiomer migration order reversal was observed between γ- and β-CD derivatives. To improve enantioseparation, buffer composition and pH, CD concentration, applied voltage, temperature and injection parameters were optimized for the alogliptin/sulfobutyl-ether-β-CD system, yielding a resolution of 8.34. RSD% 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 μg/ml respectively for each enantiomer. Calibration lines ranging from 6 to 250 μg/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 5 mM sulfobutyl-ether-β-CD in a 25 mM acetate buffer at pH 4.75 was found promising to detect 0.1% distomer in the presence of the API. This article is protected by copyright. All rights reserved.