For the first time, a new, fast and sensitive chromatographic method for the separation and determination of modafinil enantiomers was developed on chiral stationary phase with macrocyclic glycopeptide teicoplanin in the polar organic mode. The effect of the mobile phase composition and column temperature on the retention and enantioseparation were studied. The separation was performed using a Chirobiotic T column (250 × 4.6 mm, 5 μm) with methanol and triethylamine (100/0.05, v/v) as the mobile phase at a flow rate of 1.0 mL/min, and UV detection at 225 nm. The total analysis time is less than 6 min, which is faster than the previous chiral HPLC methods (total run time of 12 min). Calibration curves were linear (R2 > 0.999) over a concentration range 5–150 μg/mL for each modafinil enantiomer. Limit of detection (LOD) and limit of quantification (LOQ) for (R)-modafinil were 15 and 45 ng/mL and for (S)-modafinil were 20 and 60 ng/mL, respectively. The recoveries were in the range of 100.5-102.3% with the relative standard deviations ranging from 0.4% to 1.0% for both enantiomers. It was demonstrated that the developed method was selective, precise and robust. The validated method was successfully applied for the separation and determination of modafinil enantiomers in the real samples.