This thesis focused on optimization of separation methods for underivatized theanine enantiomers on two teicoplanin-based chiral stationary phases and separation methods for enantiomers derivatized by 9-fluorenylmethyloxycarbonyl chloride and dansylchloride. Using these optimized conditions the limits of detection and quantification were calculated. Under optimized conditions were also investigated the contents of theanine enantiomers in green tea and in selected dietary supplement containing L-theanine.