The separation and quantitation of the enantiomers and also the determination of the enantiomeric purity are now current and indispensable tasks for the pharmaceutical analysis. Among the various techniques, liquid chromatography remains the best modality owing to several advantages. High speed, sensitivity, and reproducible results make LC the method of choice in almost all laboratories. Phases that contain alpha1-acid glycoprotein as chiral selector are suitable for separation of charged and uncharged enantiomers with widely different structure. Atropine is widely used as parasympatolytic, anticholinergic and antiemetic drugs. It is one of the preferred antidote for immediate management of toxicity associated with nerve agents. Stereoselective separation was achieved with a prepacked alpha1-acid glycoprotein column without any derivatization procedure. The liquid chromatography system is coupled to mass spectrometry with an atmospheric pressure chemical ionization interface in the positive-ion mode. The chromatographed analytes are detected in selective ion monitoring after optimisation using factorial experimental design. Small amount of enantiomeric composition can be evaluated either by MS or by UV spectrometry (less than 5%).