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A sensitive high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for measuring vinorelbine was developed. A 100 mu L aliquot of plasma was spiked with deuterium-labeled internal standard and subjected to solid-phase extraction using an Oasis HLB mu-elution plate. Two microliters of the extracted samples was directly injected into LC/MS/MS. Chromatographic separation was achieved on a Capcell Pak C18 UG column (2 x 75 mm) with a gradient elution of methanol (mobile phase B) against 0.05% formic acid in aqueous 10 mM ammonium formate (mobile phase A). The LC flow rate was set to 0.28 mL/min and the gradient (solvent B concentration) was processed from 40 to 90%. In mass spectrometric detection, observation of the reaction from a double-charged precursor ion [M + 2H](2+) (m/z 390) to product ion m/z 122 provided very high sensitivity. The method was validated with a lower limit of detection of 0.2 ng/mL with 0.1 mL of plasma, and the method was used to determine the plasma pharmacokinetics of vinorelbine in dogs. Copyright (c) 2010 John Wiley & Sons, Ltd.