Hydroxypropyl-β-cyclodextrin (HPβCD) has been used successfully as a chiral additive to separate ofloxacin (OFL) enantiomers in a capillary electrophoresis system. Using electrospray-mass spectrometry (ESI–MS) it was revealed that OFL forms an inclusion complex with HPβCD at 1:1 stoichiometry. The interaction of the enantiomers with the host was further investigated by molecular modeling using molecular mechanics dockings, PM7 semiempirical calculations and molecular dynamics simulations. Calculations using PM7 semiempirical methods indicated that the separation is brought about by a large difference in the binding energies (∆∆E) of 15 kcal mol−1 between R-OFL-HPβCD and S-OFL-HPβCD inclusion complexes. S-OFL was predicted to be eluted first by the PM7 method which corroborates the experimental results. Moreover, the molecular dynamic simulations show that the formation of stable R-OFL-HPβCD is because it is more deeply inserted into the cavity of the host. The study also revealed the absence of the role of strong hydrogen bonding in the enantioseparations.