In present work, L-valine-based new chiral derivatives were prepared by introducing hydrophobic groups. The prepared derivatives were then used to prepare quinoline-based chiral reagents under mild amidation reaction and esterification reaction. Spectroscopic techniques, such as HRMS, FT-IR, 1H NMR and CHNS analysis, were used to characterize the synthesized chiral reagents. The synthesized series of reagents was then used to derivatize diastereomers of racemic mexiletine and these diastereomers were separated using the RP-HPLC (a derivatization approach of enantioseparation). The mobile phase for the analysis consisted of acetonitrile and buffer solution. The impact of modifying mobile phase pH and concentrations was optimized to separate diastereomers. The lowest energy-minimize optimized diastereomer structures, as well as the design of separation processes and elution orders, were also developed through the use of density functional theory (DFT) calculations. Following ICH guidelines, the limits of detection (0.161 ng/mL) and quantification (0.483 ng/mL) were determined, together with the retention-factor (k), selectivity-factor (α), resolution-factor (RS) and technique validation.