This paper reports the separation of two chiral antibacterial agents namely, linezolid and tedizolid using a validated high‐performance liquid chromatographic method. In the current work, glycopeptide‐based chiral column, CHIROBIOTIC® V2 (5‐μm particle size, L × I.D. 25 cm × 4.6 mm) was employed with a mobile phase containing methanol and 0.15% aq. trifluoracetic acid (75:25%, v/v) in isocratic elution approach at flow rate of 1 ml min−1. The separation condition was customized (in terms of resolution values and retention times) was carried out by changing the content of the mobile phase, column temperature, flow rate, and so on. Results showed that the chromatographic separation was achieved within 15 min and average resolution values were 4.6 and 4.8 for tedizolid and linezolid, respectively. The detection limit values were 14.85 and 14.16 ng ml−1, respectively, for tedizolid enantiomers. Further, validation of separation parameters was performed by considering the international conference on harmonization guidelines, and ultimately, the mechanism of chiral recognition was also established.