Imidazoles are well known heterocyclic compounds having important feature of a variety of medicinal agents. Studies with imidazole containing compounds are great importance in the clinical use of drugs. The presence of imidazole group in the molecules of lanoconazole (LCZ) and butoconazole (BCZ) is very important for its chemical and analytical behavior. Target analytes were efficiently separated with a Pinnacle DB Cyano column (5 mu m, 4.6 x 250 mm I.D.) and oxiconazole (OCZ) used as an internal standard. In this study, correlation between the retention factors of the solutes and pH of the mobile phase was determined at ten pH values in acetonitrile-water binary mixture. In optimization condition, quantification of these drugs were performed in human urine. The linear responses were observed over a wide concentration range (6.25.10-6-3.13.10-5 mol L-1 for LCZ and 6.32.10(-6) -2.53.10(-5) mol L-1 for BCZ) in human urine. The method were validated by determining the limits of detection (LODs, 2.01.10(-6) - 5.50.10(-7) mol L(-)1), the limits of quantification (LOQs, 6.08.10(-6) - 1.67.10(-6) mol L-1), recoveries (99.14%, 99.93%) and intra-day and inter-day relative standard deviations (RSDs, < 1%). The data presented in this report demonstrate a rapid, sensitive and accurate method for the simultaneous analysis of LCZ and BCZ in human urine for pharmacokinetic studies.