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An UHPLC–MS/MS method for the determination of paclitaxel and its metabolites in dried blood spots.Accuracy and precision were within limits of acceptance.LLOQ of 1 ng/ml without evaporation and reconstitution procedure.The assay was successfully used to support a pharmacokinetic study in mice.After being used for decades in clinical screening, dried blood spots (DBS) have recently received considerable attention for their application in pharmacokinetic and toxicokinetic studies in rodents. The goal of this study was to develop and apply a DBS-based assay for a pharmacokinetic study of paclitaxel (PTX) and its metabolites in SCID/Beige mice. A fast and sensitive UHPLC–MS/MS method has been developed for the simultaneous determination of PTX, its three metabolites (6α-hydroxy-paclitaxel, 3′-p-hydroxy-paclitaxel, and 6α,3′-p-dihydroxy-paclitaxel) and its stereoisomer 7-epi-paclitaxel. The 10 μL DBS sample was extracted with methanol for 20 min at 37 °C. After dilution of the extracts with water in a ratio of 1:1, the analytes were separated on a reversed-phase 2.1 mm I.D. column using gradient elution. The total run time was 2.5 min. The analytes were detected by use of multiple reaction monitoring mass spectrometry. The extraction recoveries of the compounds were all greater than 60%, resulting in a quantification limit of 1 ng/ml. The calibration curves ranged from 1 to 1000 ng/ml. The intra-day and inter-day imprecision (%CV) across three validation runs over four quality control levels were less than or equal to 14.6%. The accuracy was within ±11.9% in terms of relative error. The described method is advantageous in terms of its ease-of-use and speed compared to other published PTX assays. The method’s usefulness was demonstrated by applying it to a preclinical pharmacokinetic investigation of PTX and its metabolites in SCID/Beige mice with an intraperitoneal administration of 50 mg/kg Abraxane®.