The enantio-separation of three chiral sulfoxide drugs (omeprazole, pantoprazole, and lansoprazole) was achieved on Chiralpak® AS-3R column with acetonitrile‐water (65:35) at 0.5 mL/min flow and 280 nm detection within 7 min. Chiralseparation was validated properly with 9.70–14.16 and 29.39–42.91 µg.mL−1 as LOD and LOQ. The thermodynamics parameters were in the range of (-0.51 to −1,288) kJmol−1K−1(ΔG). These values confirmed chiral separation as spontaneous and thermodynamically stable. Enantiomer elution order determined on Chiralpak® IA with ethanol-hexane (95:5)at 1.0 mL/min flow at 221, 254, 270, 280,301, and 330 nm wavelengths confirmed first elution od S-enantiomer followed by R-enantiomer. This observation was also confirmed by theoretical simulations of electronic circular dichroism (ECD) calculation. The docking study was used to explain the chiral resolution of the reported drugs. As a result, a simple, fast, reliable and spontaneous and thermodynamically stable HPLC method was developed for the separation and determination of enantiomer elution order of the reported chiral sulfoxide drugs.