Voriconazole (VRC) pharmacokinetics, in particular its complex metabolism, is still not fully understood which challenges its optimal therapeutic use. To increase knowledge on the pharmacokinetics of this antifungal drug, it is essential to broaden the perspective and expand in vitro and clinical in vivo investigations in particular to aspects such as unbound plasma, target-site and metabolite concentrations. Innovative sampling approaches such as microdialysis, a minimally-invasive technique for the analysis of compound concentrations in target-site human tissue fluids, are associated with bioanalytical challenges, i.e. small sample volumes and low concentrations. Thus, a bioanalytical LC-MS/MS assay for the simultaneous quantification of VRC and its main N-oxide (NO) metabolite in human plasma, ultrafiltrate and microdialysate was developed and validated according to the European Medicines Agency guideline. Quantification was rapid, simple and feasible for clinically relevant concentrations from 5 to 5000 ng/mL in plasma and ultrafiltrate as well as from 4 to 4000 ng/mL in microdialysate. Due to the high sensitivity of the assay, only 20 microL of plasma or ultrafiltrate and 5 microL of microdialysate were required. For VRC and NO in all matrices, between-run accuracy was high with a maximum mean deviation of 7.0% from the nominal value and between-run precision was demonstrated by