We present a method for determining eight penicillin antibiotics using microemulsion electrokinetic chromatography (MEEKC). We studied how the composition of the microemulsion affected separation by modifying such parameters as the surfactant or the addition of organic solvents. The best microemulsion system consisted of 0.5% ethyl acetate, 1.2% 1-butanol, 2% Brij 35, 10% 2-butanol and 86.3% 10 mM borate buffer at pH 10. We studied the suitability of this microemulsion composition for analyzing a commercial drug. To improve the sensitivity of the method, we used the stacking technique reversed electrode polarity stacking mode (REPSM), which increased the detection limits by about 40-fold.