The main objective of this study is the use of polymeric surfactants in polyelectrolyte multilayer (PEM) coatings for the separation of the pharmaceutical substances acetylcholinesterase inhibitors (AChEIs). AChEIs are used for the treatment of Alzheimer's Disease and Myasthenia Gravis. In the open-tubular capillary electrochromatography (OT-CEC) mode, the PEM coating is evaluated using nine AChEIs. Optimal conditions are established by altering several experimental parameters such as the pH of the background electrolyte (BGE), the anionic polymer for the PEM coating, the concentration of NaCl, which is used as an additive in the polymer deposition solutions, the number of bilayers, the deposition time, and the concentration of the polymeric surfactant. 25 mM NaH2PO4 center dot H2O and 25 mM Na2HPO4 at pH 7 is used as BGE. Two bilayers of poly(diallyl dimethyl ammonium chloride) and poly(sodium N-undecanoyl L-leucinate) provide a baseline separation of all nine analytes in less than 4.5 min. Run-to-run reproducibility studies are also performed, and the relative standard deviation values of the migration times of the nine-analyte peaks are less than 2%. In addition, day-to-day, week-to-week and capillary-to-capillary reproducibilities are evaluated, and the relative standard deviation values of the electroosmotic flow are less than 2%. Finally, using the PEM coating approach, we were able to perform more than 150 runs in the same column. Neither the addition of the polymeric surfactant to the mobile phase, nor the reconstruction of the coating was necessary.