Abstract An attempt for the simultaneous separation of salbutamol (Sal) and bupivacaine (Bup) enantiomers was performed by capillary elecytrophoresis with a dual mixture of neutral cyclodextrins as chiral selector. The influence on the separation of several parameters such as buffer composition, pH, the concentration ratio of 2-hydroxypropyl-beta-cyclodextrin (HP-β-CD) to dimethyl-beta-cyclodextrin (DM-β-CD) was investigated. A better separation was obtained for Sal and Bup with the CD mixtures compared to the use of HP-β-CD or DM-β-CD alone. The best simultaneous separation of Sal and Bup enantiomers was achieved with a 20 mM HP-β-CD and 20 mM DM-β-CD at pH 2.5 in a triethanolamine (TEA)/phosphate buffer. This method-utilized chlorphenamine (Chl) as an internal standard was found to be linear in the range 0.5–100 μg/mL and 0.5–150 μg/mL for Sal and Bup enantiomers, respectively. The limits of detection for both enantiomers of Sal and Bup were 0.18 and 0.24 μg/mL, respectively. The proposed method was applied to monitor Sal and Bup enantiomers concentration change in rat blood samples obtained from a male rat after celiac doses administration 0–30 min of Sal and Bup racemate. The method could also be used to determine Sal enantiomers in a pharmaceutical aerosol.