Due to a great demand for anti-coagulant agents, two new compounds (I) and (II = thiourea derivative) chiral in nature were synthesized and characterized by UV-Visb, FT-IR and NMR techniques. The anti-coagulant activities were determined and found to be 84 and 91% for compounds I, and II by PT assay while APTT assay showed 84.5 and 90.5% activities. The enantiomers of these compounds were separated using three different chiral columns (Chiralpak-IB, Chiralcel-OJ and Chiralcel-OD-3R) with varied HPLC conditions. The separation and resolution factors for all the enantiomers were in the range of 1.25 to 3.47 and 0.48 to 1.75, respectively. The enantiomeric resolution was obtained within 12 minutes making fast separation. The reported compounds have good anti-coagulant activities and may be used as anti-coagulants in the future. Besides, the chiral separation is fast and may be used to analyze the reported enantiomers in any unknown matrices.