Summary A novel economic thin-layer chromatographic procedure for stereoselective separation of racemic mixtures of each of zopiclone and ofloxacin, and determination of their enantiomers: eszopiclone, (+)(S)-zopiclone, and levofloxacin, (−)-(S)-ofloxacin, was described. The method was based on using normal plates and hydroxy propylβ-cyclodextrin (HP-β-CD) as chiral mobile phase additive (CMPA). The spots were detected under UV lamp 254 nm, followed by densitometric measurements at 304 and 330 nm for (+)-(S)-zopiclone and (−)-(S)-ofloxacin, respectively. The mobile phase enabling successful resolution of the drugs was ethanol–acetonitrile–glacial acetic acid–diethylamine–distilled water containing 0.5% HP-β-CD (4:2:3:1:1, by volume), pH 4, for zopiclone and ethanol–acetonitrile–glacial glacial acetic acid–diethylamine–distilled water containing 0.3% HP-β-CD (4:4:3:2:1 by volume), pH 4.5, for ofloxacin at 25 ± 2°C. All variables affecting the resolution, such as concentration of different chiral selectors, temperature, and pH, were investigated, and the conditions were optimized. Furthermore, some thermodynamic parameters were calculated. The procedure provided a linear response over the concentration range of 1–4 and 2–7 μg spot−1 for determination of pure active isomers, (+)-(S)-zopiclone and (−)-(S)-ofloxacin, respectively, with acceptable precision (relative standard deviation [% RSD] <2.0). The developed method was validated and proved to be robust. The proposed method was found to be selective and accurate for the identification and quantitative determination of enantiomeric purity of the two active isomers in their drug substances and drug products.