We describe the development and validation of a novel liquid chromatography assay (HPLC/PDA) for simultaneous quantification of voriconazole, itraconazole, and posaconazole, as well as some of their major metabolites, voriconazole N-oxide and hydroxy-itraconazole, in human serum. Analytes were detected using a PDA system that allows the characterization of the specific UV spectra of each compound. The assay exhibited linearity between 0.25-16 mg/L for all the compounds tested. The accuracy and within- and between-day precision of the assay were in acceptable ranges. We successfully quantified the azoles and some of their metabolites in a collection of clinical samples collected from treated patients. The method also allows assessing the metabolic rate of several azoles being useful to predict the metabolic profile of a particular patient and to anticipate toxicity or efficacy during the treatment.