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Graphical abstract Figure. No Caption available. HighlightsCD‐NACE enantioseparation of sutezolid, linezolid and its precursor.Enantiospecific host:guest interactions by spectroscopic approach, FT‐IR, NMR.Molecular modelling were investigated to provide information about complex formation. ABSTRACT A method for the enantioseparation of sutezolid, the next analogue after linezolid and tedizolid, belonging to the truly new class of antibacterial agents, the oxazolidinones, was developed based on non‐aqueous capillary electrophoresis (NACE), using a single isomer of cyclodextrins as a chiral pseudophase. During the experiment, the enantioseparation of sutezolid together with its predecessor, linezolid, both weak base antibacterial agents, was evaluated using anionic single‐isomers of cyclodextrins from hydrophilic, up to hydrophobic: heptakis‐(2,3‐dihydroxy‐6‐sulfo)‐&bgr;‐cyclodextrin, heptakis‐(2,3‐diacetyl‐6‐sulfo)‐&bgr;‐cyclodextrin (HDAS‐&bgr;‐CD), as well as heptakis‐(2,3‐dimethyl‐6‐sulfo)‐&bgr;‐cyclodextrin (HDMS‐&bgr;‐CD), respectively. Based on the observed results, the cyclodextrins, HDAS‐&bgr;‐CD and HDMS‐&bgr;‐CD which carry the acetyl and methyl groups at the C2 and C3 positions, respectively, provided the baseline separation of sutezolid enantiomers. However, HDMS‐&bgr;‐CD led to a reversal of enantiomer migration order (EMO) in comparison to HDAS‐&bgr;‐CD. Instead, enantiomers of linezolid were separated only by HDMS‐&bgr;‐CD. During the experiments, different organic solvents and their mixtures in various ratios were tested. The selectivity and separation efficiency were critically affected by the nature of the buffer system, the type of organic solvent, and the concentrations of trifluoroacetic acid (TFA) in the NACE buffer system. Focusing on the desired EMO in which the eutomers (S)‐sutezolid and (S)‐linezolid migrated last, the highest enantioresolution using the NACE method was achieved at normal polarity mode with 45 mM HDMS‐&bgr;‐CD dissolved in MeOH/ACN (85:15, v/v) containing 200 mM TFA/20 mM ammonium formate. Moreover, infrared spectroscopy, NMR and molecular modelling were investigated to provide information about complex formation.