A high-performance liquid chromatographic (HPLC) method is described for direct separation of the enantiomers of metipranolol and its principal degradation product and main metabolite, desacetylmetipranolol. Separations are performed on a chiral stationary phase of cellulose tris-3,5-dimethylphenyl carbamate (Chiralcel OD), with hexane-propan-2-ol-diethylamine elution and UV detection at 278 nm. The method affords identification and determination of the optical purity of the bulk drug and its formulated ophthalmic products.