The enantioseparation and resolution mechanism of mandelic acid (MA), 4-methoxymandelic acid (MMA), and 4-propoxymandelic acid (PMA) were investigated by reversed-phase high-performance liquid chromatography (HPLC) with 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) as a chiral mobile-phase additive and molecular dynamics simulation. The suitable chromatographic conditions for the enantioseparation of MA, MMA, and PMA were obtained. Under the selected chromatographic conditions, these enantiomers could achieve baseline separation. The results of thermodynamic parameter analysis revealed that the main driven forces for the enantioseparation of MA, MMA, and PMA could be van der Waals forces and hydrogen-bonding interactions and the chromatographic retention of these chiral compounds was an enthalpy-driven process. The results of the molecular simulation revealed that their chiral resolution mechanism on HP-ß-CD was responsible for the formation of inclusion complexes of enantiomers with HP-ß-CD with different conformations and binding energies. And the binding energy of HP-ß-CD with (S)-isomer was larger than that with (R)-isomer, which is consistent with the experimental results of the first elution of (S)-isomer. Additionally, it is also confirmed that the interaction energies included the van der Waals energy (∆Evdw ), electrostatic energy (∆Eelec ), polar solvation energy, and SASA energy (∆Esasa ), and the separation factor (α) was closely connected with the disparity in the binding energies of optical isomers and HP-ß-CD complexes. Meanwhile, from molecular dynamics simulation, it can be found that the ∆(∆Ebinding ), (∆(∆Ebinding ) = ∆Ebinding,R - ∆Ebinding,S ) value was in order of MA-HP-ß-CD complex > MMA-HP-ß-CD complex > PMA-HP-ß-CD complex, which was consistent with the order of Δ(ΔG) values obtained from van't Hoff plot. This indicated that the molecular dynamics simulation has predictive function for chiral resolution.