We have developed and validated a new and reliable gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method with a diode array detector (DAD) for the simultaneous separation and determination of 23 frequently prescribed selected drugs belonging to different therapeutic groups in human urine samples. For the drugs listed below, this method of analysis for human urine was also successfully applied to determine urine concentrations of these drugs in samples from treated patients: enalapril (ENA), paracetamol (PAR), sotalol (SOT), dipyrone (DIP), vancomycin (VAN), captopril (CAP), fluconazole (FLU), cefazolin (CEF), metoprolol (MET), aspirin (ASP), ticlopidine (TIC), prednisolone (PRE), propranolol (PRO), digoxin (DIG), sildenafil (SIL), furosemide (FUR), dexamethasone (DEX), carvedilol (CAR), ketoprofen (KET), nifedipine (NIF), terbinafine (TER), acenocoumarol (ACE) and spironolactone (SPI). Separation of the analytes was achieved by RP-HPLC-DAD with a mobile phase composed of acetonitrile, methanol and 0.05% trifluoroacetic acid in water using a gradient elution program. Good linear relationships over the investigated concentration ranges were observed with values of r 2 higher than 0.998 for all of the drugs. The intra-day and inter-day precisions of this method were evaluated with RSD values less than 4.26 and 5.42%, respectively. The relative recoveries of the 23 investigated compounds ranged from 93.60 to 106.00% with RSD values less than 4.46%. An expanded uncertainty budget was constructed for all investigated drugs in human urine samples.