Voriconazole is a novel broad-spectrum antifungal agent. We developed an on-line LC-LC-MS-MS method for fully automated and direct analysis of voriconazole in raw human serum. After injection of human serum size-selective sample fractionation and analyte extraction was achieved using an extraction column (25 mm × 4 mm) packed with a restricted access material (RAM, LiChrospher® ADS C 8, 25 μm). On-line transfer of voriconazole from the extraction column was followed by chromatography separation on a C 18 column. Detection was done by ESI-MS-MS. The total analysis time was 13 min, managed by parallel extraction and chromatographic separation. This LC-MS assay was fully validated. The lower limit of quantification was 0.05 μg/ml. The automated inline extraction of voriconazole described here eliminates the need for difficult and time-consuming sample pre-treatment. Other advantages of the new method are that only a small quantity (5 μl) of serum is needed and that the method is very specific. © 2004 Elsevier B.V. All rights reserved.