An enantioselective CE assay for the simultaneous determination of the enantiomeric purity and of related substances of etomidate has been developed and validated using a binary chiral selector system employing 30 mg/mL beta-CD and 4.6 mg/mL sulfated-beta-CD in a 150 mM potassium phosphate buffer, pH 2.1. The method was validated with respect to specificity, range, linearity, LOQ and LOD, precision and accuracy. The assay allowed the detection and determination of related substances including (S)-etomidate at the 0.05% w/w level, the reporting threshold as defined by the International Conference on Harmonisation guidelines as well as the European Pharmacopoeia. Robustness testing was carried out by an "Augmented Plackett-Burman" design. Quantitation of the compounds was performed by calibration graphs with respect to lidocaine hydrochloride as internal standard and by peak area normalization, the procedure usually applied by pharmacopoeias. Although data obtained from the calibration graphs constructed with the aid of the internal standard were more accurate based on compound recovery, peak area normalization may also be used without significant loss of accuracy and precision.