The purpose of this paper was to study at the molecular level the enantioseparation mechanism of 4-hydroxypropranolol (4-OH-Prop) with carboxymethyl-β-cyclodextrin (CM-β-CD) using a sequential methodology which included molecular dynamics simulations (MD), and Parametric Model 3 (PM3) semiempirical and density functional theory (DFT) calculations. A systematic structural analysis indicated that hydrogen bonds formed between the host and guests play a major role in the complex stabilization. The inclusion complex (+)-(R)-4-OH-Prop/CM-β-CD showed three strong intermolecular hydrogen bonds. Moreover, the guest inclusion process made from a wider CD rim presented lower energies (interaction and Gibbs free energy) in comparison to the inclusion made by a narrower CD rim in both gas and aqueous phases. This difference in energies of drug/CM-β-CD inclusion complexes is probably a measure of chiral discrimination, which results in the separation of the enantiomers and the distinct separation factors as observed in previous experimental findings. Comparing the experimental results of the separation of 4-OH-Prop enantiomers by capillary electrophoresis (CE), the proposed theoretical model demonstrated good capability to predict chiral separation of 4-OH-Prop enantiomers as well as the qualitative estimative of chiral recognition mechanism.