This study aims to synthesize hesperidin oxime from hesperidin, chiral separation by HPLC method and comparative study of two enantiomers as anti-inflammatory agents by docking them with Proto-oncogene serine/threonine-protein kinase Pim-1 using MOE software (4.2). The structure of the synthesized product was validated through UV, IR,1H-NMR, and13C-NMR spectroscopic techniques. Hesperidin-oxime was separated chirally in normal phase mode employing polysaccharide-based chiral stationary phases, namely Chiralcel® OD-H, Chiralcel® OD-RH, and CHIRALPAK® AD-3R, under both normal and polar organic phase modes. Molecular docking shows that S- hesperidin oxime gave better binding affinity towards pim-1 compared to R- hesperidin oxime.