Capillary electrophoresis is a powerful separation technique for analysis of proteins and peptides, with benefits like low consumption of reagents, solvents and sample. The separation efficiency and resolution can be deteriorated by adsorption of analytes to the inner capillary wall, though. Many methods to circumvent this obstacle have been reported, including background electrolyte addition of surfactants that aggregate as protective coatings at the wall. In this work, anionic suberin surfactant was used together with the cationic surfactant cetyltrimethylammonium bromide (CTAB) for analysis of trypsin digested lysozyme as a model sample. Suberin fatty acids were extracted from birch bark, which is a side-stream product originating from pulp and paper waste streams. Different adjustments of the solvent extraction protocol, and the method to neutralize the suberin fatty acids to obtain surface active sodium salts were evaluated regarding number of peaks observed, separation repeatability, and analysis time. The influence of background electrolyte pH was also studied. The potential of the surface-active sodium salts of suberin fatty acids as an additive enhancer in combination with CTAB is illustrated by excellent repeatability, especially at lower pH values. The number of peaks observed was also higher at lower pH, while the analysis time was shorter.