Six insulin analogues with high degree of chemical similarity were separated using Capillary Electrophoresis (CE). In order to improve the method performance, optimization and development the Design of Experiment (DoE) approach has been used and this strategy provided related statistical models. This methodology delivered the information regarding the influence of main factors in the method development and explained the interaction of relevant factors in terms of shortening the analysis time. The response surface methodology (RSM) design employing Central Composite Face Centered (CCF) Design analyzed the effect of the most influencing factors, including background electrolyte pH and concentration, applied voltage and temperature. This study demonstrated that the approach of analyzing the influence of each parameter in the migration behavior of analyzed peptides was capable to assess the best electrophoretic conditions for the separation of insulin analogues.