Determination of optical isomers is based on some molecule properties to have one or more chiral asymmetrical carbon center. Enantiomers can be resolved by high performance liquid chromatography (HPLC) with a chiral mobile phase additive, by change into diastereoisomers by derivatization or by separation on chiral column. We retained the last technique for the separation of seven beta-blockers (atenolol, alprenolol, labetalol, nadolol, oxprenolol, pindolol and propranolol) using an alpha 1-AGP column. Results indicate that the mobile phase was specific to each beta-blocker. The wavelength chosen was lambda = 225 nm and the flow rate was 0.9 ml/min. A part from that, determinations of isomers of propranolol and atenolol in different fluids biologicals were also achieved.