The applicability of capillary zone electrophoresis for the simultaneous determination of cefamandole and probenecid in body fluids has been studied using salicylic acid as an internal standard. A 10 mM disodium tetraborate solution (pH 8.0) was used as a running buffer and the UV wavelength was set at 230 nm for monitoring the analytes at a separation voltage of 18 kV. Under the optimized conditions, the analytes can be effectively separated in 6 min. The standard curves of cefamandole and probenecid showed good linearity in the range of 10–200 and 5–110 μg mL⁻¹, respectively, with correlation coefficients r > 0.999. The detection limits (S/N = 3) of cefamandole and probenecid were 2.7 and 1.1 μg mL⁻¹, respectively. Intra- and inter-day analytical precisions (relative standard deviation, RSD) of peak area and migration time for the two medicines were both less than 2%. The recoveries at three spiked levels of cefamandole and probenecid from urine and serum samples were in the range of 96.23–105.1% with RSD values of 0.55–2.36%. The proposed method was tested for clinical application by the simultaneous determination of cefamandole and probenecid in human urine and serum with satisfactory results.