Enantiomeric resolution of atenolol was achieved on the HPLC vancomycin macrocyclic antibiotic chiral stationary phase Chirobiotic V. The polar ionic mobile phase consisted of methanol-glacial acetic acidtriethylamine (100 + 0.025 + 0.75, v/v/v) at a flow rate of 0.8 mL/min. Fluorescence detection at 2751305 nm for excitation and emission, respectively, was used. Plasma samples were purified using SPE on Oasis HLB cartridges. The calibration curves in plasma were linear over the range of 5-400 ng/mL (r = 0.999) for each enantiomer with an LOD of 1.0 ng/mL. The proposed method was validated in compliance with International Conference of Harmonization guidelines in terms of linearity, accuracy, precision, LOD, LOQ, and selectivity. The overall recoveries for S-(-)- and R-(+)-atenolol enantiomers from plasma were 95.0-99.5%; RSD ranged from 2.5 to 3.3%. The developed method was applied for the trace analysis of atenolol enantiomers in plasma and for the pharmacokinetic investigation of atenolol enantiomers in mouse plasma.