The present paper deals with the enantiomeric separation of six antihistaminic enantiomers by affinity electrokinetic chromatography (AEKC)-partial filling technique using human serum albumin (HSA) as chiral selector. A multivariate optimization approach of the most critical experimental variables in enantioresolution, running pH, HSA concentration and HSA plug length (SPL) was carried out since there are interactions between variables that could not be considered in an univariate optimization. The estimated and experimental resolution values obtained for antihistaminic enantiomers varied from 1.13 (for orphenadrine) to 2.15 (for brompheniramine). The optimum experimental conditions for enantioresolution of each compound were: brompheniramine, pH 8.5, [HSA] 180 microM, SPL 180 s; chlorcyclizine, pH 6.5, [HSA] 180 microM, SPL 150 s; chlorpheniramine, pH 8.25, [HSA] 160 microM, SPL 150 s; hydroxyzine, pH 7.0, [HSA] 180 microM, SPL 150 s; and orphenadrine, pH 7.8, [HSA] 160 microM, SPL 150 s. pH and the quadratic term of pH seem to be the most critical factors that determine enantioresolution of antihistamines. The validity of the developed methodologies to enantiomeric quality control of antihistamines in pharmaceutical formulations is demonstrated analyzing the content of brompheniramine, chlorpheniramine and hyroxyzine enantiomers in commercially available pharmaceutical formulations containing racemic mixtures of compounds. Resolution, accuracy, reproducibility, cost and sample throughput of the proposed methodologies make them suitable for quality control of the enantiomeric composition of antihistamines in pharmaceutical preparations.