In DNA analysis by capillary electrophoresis with polymer solutions there are many variables that can be optimized. However, electric field strength, polymer solution concentration and temperature of analysis are the most relevant ones. These are the variables most responsible for the fragment resolution and analysis time. Optimization of such parameters can be obtained simultaneously using chemometric techniques, reaching the optimum working conditions with few experiments. In this work, we have studied the influence of the sample composition and electrokinetic injection conditions in the reproducibility and the quality of the DNA separation results. A simplex optimization has been carried out and the optimum condition was reached with nine experiments.