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A simple, fast, and low-cost method was developed and validated for the separation and determination of neostigmine metilsulfate (Neo), naphazoline hydrochloride (Nap), chlorphenamine maleate (Chl), pyridoxine hydrochloride (Pyr), and dipotassium glycyrrhizinate (Dip) by capillary electrophoresis (CE) for the first time. The separation was achieved in 7 min with 10 mM borate of pH 10.0, ultraviolet detection at 205 nm, and 25 kV applied voltage. The proposed method demonstrates good linearity with r2between 0.9995 and 0.9998. The repeatability was in the range of 0.23–1.96% with migration time and 0.81–3.46% with peak area for the five components. The limits of detection were 0.10, 0.02, 0.08, 0.17, and 2.44 µg mL−1for Neo, Nap, Chl, Pyr, and Dip, respectively. The method was successfully applied to the quantitation of the five components in a commercial eye drops with recoveries in the range of 99.01–101.26%. Furthermore, a comparison of the results obtained by CE was made with those obtained by high-performance liquid chromatography (HPLC) described in Chinese Pharmacopoeia. The performance of the proposed method was comparable, and the quantitative results were in good agreement with those by HPLC, while the CE method has the advantages of shorter analysis time and lower cost than HPLC. [ABSTRACT FROM AUTHOR]