A new analytical methodology using EKC with a CD as chiral selector was developed enabling the simultaneous enantiomeric separation of two chiral neurotransmitters (epinephrine and norepinephrine). Different experimental conditions (type and concentration of the chiral selector, nature and concentration of buffer and separation voltage) were optimized. The use of 10 mM carboxyethyl-beta-CD in 100 mM Tris-phosphate buffer (pH 5.0) with an applied voltage of 30 kV and a temperature of 15 degrees C allowed the separation of epinephrine and norepinephrine enantiomers with high chiral resolution (Rs > 2.5). Moreover, after evaluating the analytical characteristics of the method, it was applied to the quantitation and purity testing Of L-epinephrine as active pharmaceutical ingredient in pharmaceutical formulations where D-epinephrine and L/D-norepinephrine can be present as impurities.