The present work investigated the non-aqueous CE (NACE) method for the separation of β-agonists (i.e., clenbuterol, salbutamol and terbutaline). Two detectors, diode array detector (DAD) and mass spectrometer (MS), had been used for the etection of the β-agonists. Non-aqueous media was selected to improve the selectivity and separation efficiency of CE. Additionally, non-aqueous solvent can improve compatibility with a MS. The initial NACE–DAD condition was developed by using factorial design to optimize the experimental factors including background electrolyte concentration, organic solvent, injection time, voltage and temperature.