Abstract: Hydroxypropyl-β-cyclodextrin (HP-β-CD) was chosen as a chiral selector to be added to the mobile phase to achieve separation of enantiomers of cephalothin by UHPLC. Agilent Poroshell 120 SB-C₁₈ (2.1 mm×100 mm, 2.7 μm) chromatographic column was used as stationary phase. A mixture of acetonitrile and 12.0 mmol·L^-1 HP-β-CD solution (pH 7.2), mixed in the ratio of 80 to 20, was used as mobile phase with a flow rate of 0.25 mL·min^-1. Under the chosen conditions, good baseline separation of the enantiomers of cephalothin was attained, and giving values of RSD's obtained from 6 successive determinations no more than 2.3%.