Experimental conditions have been studied in order to improve the sensitivity for the analysis of diuretics and probenecid in urine samples by high-performance liquid chromatography with ultraviolet detection. Sample clean-up and chromatographic parameters have been optimized to obtain a suitable sensitivity for the detection or quantification of each diuretic using an HP-Hypersil ODS-C18 column (5 microns, 250 mm x 4 mm I.D.), taking into account the pharmacological properties of each compound. The reliability of this method was tested by analysing urine samples after a minimum single-dose administration of chlorthalidone and probenecid.