Panthenol is a biologically active compound closely related to vitamin B5 (pantothenic acid). This work deals with the separation of panthenol enantiomers using high performance liquid chromatography. Different types of chiral stationary phases (β-cyclodextrin, isopropyl carbamate cyclofructan 6, amylose tris(3,5-dimethylphenylcarbamate)) were tested in normal phase separation mode. Effect of mobile phase composition on the resolution and retention of enantiomers was studied. Two types of detectors, low-wavelength UV and polarimetric, were used. The optimal chromatographic system includes a chiral stationary phase based on amylose and a mobile phase of hexane/ethanol (60/40, v/v) where the resolution of enantiomers reached the value Rs = 2.49. Suitable chromatographic conditions were applied for the determination of panthenol enantiomers in samples of pharmaceutical preparations with the obtained recovery of more than 92 %. Linearity of the high performance liquid chromatography method with spectrophotometric detection was from 1.0 × 10−3 to 1.3 mg mL−1 (R2 = 0.998), with the limit of detection of 0.3 × 10−3 mg mL−1 for both enantiomers.