The objective of this study was to develop a fast and robust method for the quantitation of the antifungal drug anidulafungin in human plasma samples by generic two-dimensional liquid chromatography (online-SPE/reversed phase LC) coupled to a tandem-quadrupole mass spectrometer (LC-online SPE-MS/MS). Online SPE was performed using an Oasis HLB cartridge column and for reversed-phase chromatography a Nucleodur Gravity C18 column was used. A 100 μL aliquot of human plasma was extracted with 200 μL of 80:20 MeOH–0.2 M ZnSO4 (v/v) as precipitation reagent containing ascomycin as internal standard (IS). The supernatant was directly injected for analysis. The total run time was 4.5 min. Anidulafungin and ascomycin were detected in the positive ionization mode. The method performance data for anidulafungin, such as limit of detection (0.013 µg/mL), lower limit of quantitation (0.04 µg/mL), linearity (R2 = 0.9999) and concentration range (0.04–10 µg/mL) were ascertained. Intra- and inter-day precisions were ≤6.6% and intra- and inter-day accuracies were 98.5–101.0 and 100.0–102.5%, respectively. The assay was successfully applied for quantitation of anidulafungin in patient plasma samples. Copyright © 2011 John Wiley & Sons, Ltd.