Nonaqueous capillary electrophoresis (NACE) was successfully applied to the resolution and the determination of salbutamol enantiomers in urine samples using heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-cyclodextrin (HDAS-beta-CD). After optimization of the electrophoretic parameters, namely the background electrolyte (BGE) composition and the HDAS-beta-CD concentration, salbutamol enantiomers were completely resolved using a BGE made up of 10 mM ammonium formate and 15 mM HDAS-beta-CD in methanol acidified with 0.75 M formic acid. Isoprenaline was selected as internal standard. Solid-phase extraction (SPE) was used for sample cleanup prior to the CE separation. Different sorbents involving polar, nonpolar interactions or dual retention mechanisms were evaluated and extraction cartridges containing both nonpolar and strong cation-exchange functionalities were finally selected. Salbutamol enantiomers recoveries from urine samples were determined. The method was then successfully validated using a new approach based on accuracy profiles over a concentration range from 375 to 7500 ng/mL for each enantiomer.