A fast capillary zone electrophoresis method for the simultaneous analysis of glibenclamide and its impurities (IA and IB) in pharmaceutical dosage forms was fully developed within a quality by design framework. Critical quality attributes were represented by IA peak efficiency, critical resolution between glibenclamide and IB, and analysis time. Experimental design was efficiently used for rapid and systematic method optimization. A 3(5)//16 symmetric screening matrix was chosen for investigation of the five selected critical process parameters throughout the knowledge space, and the results obtained were the basis for the planning of the subsequent response surface study. A Box-Behnken design for three factors allowed the contour plots to be drawn and the design space to be identified by introduction of the concept of probability. The design space corresponded to the multidimensional region where all the critical quality attributes reached the desired values with a degree of probability π ≥ 90%. Under the selected working conditions, the full separation of the analytes was obtained in less than 2 min. A full factorial design simultaneously allowed the design space to be validated and method robustness to be tested. A control strategy was finally implemented by means of a system suitability test. The method was fully validated and was applied to real samples of glibenclamide tablets. Graphical Abstract Identification of sequence variants in IgG Fc mixtures.