Itraconazole is a new triazole antifungal agent. Active orally, this drug is effective against a wide range of fungal pathogens that includes Aspergillus species, and its use in leukemic and AIDS patients is currently on the increase. Oral itraconazole absorption presents up to threefold interindividual variation in man and is reduced in AIDS patients. Consequently an individual itraconazole adjusting dosage is necessary to ensure adequate clinical antifungal activity. Itraconazole undergoes extensive metabolism and the main isolated metabolite, hydroxyitraconazole, is found in plasma at concentrations 2-3-fold higher than parent drug and presents in vitro the same antifungal activity. At present, despite the contribution of this metabolite to the overall activity of the drug, no well-documented assay was reported in the literature for the codetermination of itraconazole and hydroxyitraconazole in plasma. Due to the wide variety of coadministered drugs to patients receiving itraconazole, the purpose of the developed method was to obtain a specific assay sensitive enough for itraconazole therapeutic monitoring. Therefore, a three-step liquid-liquid extraction procedure following by reversed-phase chromatography and spectrofluorimetric detection was performed. This assay allowed determination of 20 ng/ml of both itraconazole and its active metabolite with an acceptable precision using a 0.5-ml plasma sample; no analytic interference was encountered from 45 coadministered drugs tested.