A practical approach for the evaluation of binding constants with affinity capillary electrophoresis (ACE) is presented using the different linear and nonlinear regression methods. The influence of the sample concentration on the different obtained curves is depicted and it is shown that the different representations should always be compared. The well-known strong molecular interaction between the macrocyclic antibiotic vancomycin and a (D)-Ala-(D)-Ala terminating peptide is used as model in this study.