The combination of some drugs can enhance pharmacodynamics. Levofloxacin (LOFX), cefazolin (CFZL), and cefminox (CMNX) can be combined and administered perorally or intravenously and are excreted mainly into the urine. A new ion-pair RP-HPLC method was developed, for the first time, for effective separation and simultaneous determination of the three drugs in human urine. The analytical conditions were investigated and optimized. Under optimal conditions, the three drugs can be separated effectively. Good linearity was achieved in 0.2450 mu g/mL for LOFX, 151500 mu g/mL for CFZL, and 0.660 mu g/mL for CMNX, with the correlation coefficients(r) of >0.999. The LOD values for LOFX, CFZL, and CMNX were 0.08, 3.75, and 0.10 mu g/mL, respectively. The recoveries ranged from 110 to 115% with the RSD of 2.43.9% for LOFX, from 101 to 115% with the RSD of 2.23.2% for CFZL, and from 94.7 to 103% with the RSD of 1.03.2% for CMNX. The proposed method is effective, accurate, and provides the sensitivity and linearity necessary for analysis of the studied drugs in human urine at clinically relevant concentrations.