A sensitive and effective method for the detection and enantioseparation of β-blockers was developed. Three quinoline-based active chiral reagents were prepared followed by an amide and ester formation reaction. The synthesized active chiral reagents were characterized with spectroscopic techniques such as NMR, FT-IR, UV, HRMS and elemental analyzer. The active reagents were used in the diastereomerization of the racemic β-blockers (propranolol, metoprolol and carvedilol). Synthesized diastereomeric pairs were then subjected to RP-HPLC for chromatographic separation. The eluting phase, which consists of a buffer solution with acetonitrile, was used for analysis. The separation analysis results were optimized in the presence of variables such as eluting phase ratio, concentration and pH. Additionally, the separation process, elution order and stable conformer of diastereomers were studied by developing minimized energy structures of prepared diastereomeric pairs using DFT.