We separated the enantiomers of atropine, a main ingredient of Scopolia extract and Scopolia Rhizome, by capillary electrophoresis. The best conditions for chiral separation were investigated based on the concentration and type of cyclodextrin (CD) used, the pH, the concentration of the electrolyte solution and the capillary temperature. Good resolution of D- and L-hyoscyamine (atropine) was achieved in 100 mM phosphate buffer (pH 2.5) containing 30 mM trimethyl-bcyclodextrin (TM-b-CD) as the chiral selector. The calibration curves showed good linearity in the range of 10–200 mg/ml (r>0.99) for D-hyoscyamine, L-hyoscyamine and scopolamine. We could analyze atropine from the samples of crude drugs and pharmaceutical preparations according to the procedures described in The Japanese Pharmacopoeia (Thirteenth Edition).