WHO is searching most active antibiotics due to the bacterial resistance problem. The activities of the racemic antibiotics may be augment by preparing optically active antibiotics by the chiral separation. Chiral separation of potential antibiotics such as cefotaxime and ofloxacin was studied using amylose-based packing chiral stationary phases (CSPs) such as Chiralpak IA and Chiralpak IG. Supercritical fluid chromatography (SFC) was employed to carry out this study. Both immobilized CSPs such as Chiralpak IA and IG have sown remarkable selectivity for the reported drugs by using SFC. The values of retention factor (k) for ofloxacin enantiomers were 9.63 and 11.81, followed by 2.94 and 5.96 for cefotaxime enantiomers. The values of separation factor (α) for both the reported drugs were 1.22 and 2.03, respectively Similarly, the values of resolution factor (Rs) for both the enantio-selective drugs were 1.49 and 2.06, separately and respectively. The chiral recognition mechanism was developed and it was observed that the π-π interactions are playing a major role. The developed method is effective, reproducible, eco-friendly, and may be used to discriminate the enantiomers of the reported drugs in any sample.