A micellar electrokinetic chromatography method is described for the simultaneous determination of hyoscyamine, scopolamine and littorine. The separation buffer consisted of a 30 mM borate-phosphate buffer at pH 8.7, in the presence of 40 mM sodium dodecyl sulfate and 16.5 % acetonitrile. The method is applied to the determination of these alkaloids in various hairy root and plant extracts.