Serum concentration of seven antifungal agents, amphotericin B, 5-flucytosine, ketoconazole, fluconazole, itraconazole, miconazole and econazole were assayed using a single step sample preparation and an isocratic High Performance Liquid Chromatography (HPLC) procedure based on three mobile phases of similar components. Our method was simple, flexible and rapid, the assays being completed within half an hour. The method showed high reproducibility, good sensitivity with detection limits of 0.078 to 0.625 mg/L except for miconazole and econazole, and high recovery rates of 86-l05%. Out of 24 therapeutic agents tested only aztreonam and trimethoprim were found to interfere with the assay of 5-flucytosine and fluconazole respectively, using this protocol. HPLC assay should be useful in the clinical laboratory for monitoring patients on antifungal therapy.