In treating the diseases of the cardiovascular system, most of the 50 used β-blocking agents are aryloxy-propanolamine derivatives, containing chiral center. Tipically, the S(-) enantiomers are more active than the R(+) enantiomers. For this reason some of them (for example timolol) are used as a single enantiomer. For the determination of the enantiomeric purity of timolol the recent edition of the European Pharmacopoeia  prescribes a HPLC method using chiral stationary phase. There are several chiral chromatographic methods for the determination of enantiomeric purity of medicinal substances in the pharmaceutical bibliography. In our studies we used several (16) β–blockers. For the enantioseparation of these compounds we applied the method of liquid chromatography, using Chirobiotic T (50 × 4,6 mm, 5 μm, containing teicoplanin) chiral stationary phase. In case of 15 compounds chiral interaction was observed. Baseline separation of 6 compounds was obtained.