Abstract A gradient normal-phase high-performance liquid chromatography (NP-HPLC) method has been developed for the determination of impurities in α-cyclodextrin. The method is selective for six linear sugars and several late-eluting unidentified impurities, as well as for α-, β- and γ-cyclodextrins. The HPLC system utilizes mobile phases consisting of acetonitrile–water (A, 64:36; B, 35:65), a 5-μm YMC Polyamine II column (250×4.6 mm I.D.), and pulsed amperometric detection. Experiments were conducted to determine the optimal mobile phase concentrations, establish the precision of the method, and demonstrate linearity and determine relative response factors for select sugars. The method was applied to select lots of commercial-grade α-cyclodextrin from two different manufacturers. The method is novel in that it offers gradient elution of strongly-retained impurities at ambient column temperatures and pulsed amperometric detection.