A novel capillary zone electrophoresis (CZE) method is, for the first time, developed for effective separation and simultaneous determination of ceftriaxone sodium and levofloxacin in human urine. The electrophoresis conditions were investigated and optimized. A 25 mM disodium tetraborate buffer adjusted with sodium hydroxide or phosphoric acid to pH 9.2 was used and the peak area was determined with UV detection under 20 kV separation voltages. Under optimized conditions, the two drugs can be separated effectively. Good linearity of quantitative analysis was achieved in the range of 10 – 500 μg mL – 1 for ceftriaxone sodium and 25 – 500 μg mL – 1 for levofloxacin with the correlation coefficients (r) of > 0.999. The limits of detection (LOD) for ceftriaxone sodium and levofloxacin were 2.5 and 7.5 μg mL – 1, respectively. Their average recovery from human urine was 99.15 % with the RSD of 0.65 – 1.54 % for ceftriaxone sodium, and 99.57 % with the RSD of 0.2 – 2.0% for levofloxacin. The proposed method is simple, rapid and accurate, and provides the sensitivity and linearity necessary for selective and simultaneous analysis of the test drugs in human urine at clinically relevant concentrations.