Caspofungin (MK-0991; L-743,872) is the first representative of a new important class of antifungal agents, the glucan synthesis inhibitors. To the authors' best knowledge, to date only one high-performance liquid chromatography (HPLC) method has been published for the determination of caspofungin in serum. Severe difficulties with sorption were described. We developed a new method which addresses these difficulties using an advanced column-switching technique for fully automated analysis of caspofungin in serum without any pre-treatment. Extraction was performed automatically inline, using a diol column, followed by chromatography on a CN column. Detection was performed by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) with isolation and fragmentation in the positive ion mode. Total analysis time was 30 min. Detection of caspofungin was achieved by retention time, isolation and fragmentation of the double positively charged caspofungin ion. This LC/MS assay was validated for between-run accuracy (max. 110%) and precision (max. CV 16.1%). The lower limit of quantification was 0.2 μg/mL. The analytical method with fully automated inline extraction of caspofungin described here removes the need for difficult and time-consuming sample pre-treatment. Sorption of caspofungin is not of importance. Additional advantages of the new method are that only a small quantity of serum (5 μL) is needed and that the method is very specific. Copyright © 2004 John Wiley & Sons, Ltd.