A method for the enantioseparation of linezolid, the first compound of a truly new class of antibiotics—the oxazolidinones, was developed. The elaborated method of linezolid enantiomers separation was successfully performed using an anionic single-isomer cyclodextrin—heptakis-(2,3-diacetyl-6-sulfato)-β-cyclodextrin (HDAS-β-CD) as a resolving agent with the help of the charged resolving agent migration model (CHARM model). The best results were obtained with 27.5mM HDAS-β-CD dissolved in 50mM borate buffer, pH 9.0, 15°C, normal polarity. The facile strategies for the reversal of the enantiomers elution order are also described. Afterwards, the optimized method was validated in terms of sensitivity, linearity, accuracy and precision.