An improved HPLC procedure for separating ciprofloxacin and its four metabolites, M1, M2, M3, M4 in urine, was developed. The procedure used is reversed phase chromatography, the stationary phase being the C18-column, followed by quantification using a UV detector. The mobile phase is a mixture of isopropanol, acetonitrile, tetrabutylammonium bromide (TBABr), heptanesulfonic acid (HSA), and 0.05% triethylamine, (adjusted to pH=3.0 with phosphoric acid.) In the process of determining the optimum separation condition, the effect of each ion-pairing reagent on the retention of all analytes was investigated and explained as well. This method involves an off-line solid phase extraction, using C18Sep-Pak cartridges for ciprofloxacin and its metabolites in urine. The desorption condition is investigated by using different kinds of organic solvents, or a mixture of organic solvents and phosphate buffer, adjusted to different values of pH. At the optimum desorption condition, all analytes show a recovery rate over 90%. Based on this optimum condition, this method was applied to pharmacokinetic study of solutes in urine sample.