DV1 is a potent and selective D-peptide antagonist of CXCR4 and being developed as a novel drug candidate molecule. For preclinical pharmacokinetic study of DV1, we established an efficient and reliable liquid chromatography coupled to tandem mass spectrometric (LC–MS/MS) method for the assay of DV1 in rat plasma. Plasma samples were acidified by formic acid and then their protein content precipitated by acetonitrile. Sample separation was processed with a C18 column (4.6 mm × 100 mm, 5 μm) and washed by a water-acetonitrile gradient mobile phase containing 0.1% (v/v) formic acid at a flow rate of 0.4 mL/min. The mass spectrometer was operated in the multiple reaction monitoring mode and positive electrospray ionization. The assay had a good linearity over the range of 10–10000 ng/mL (r>0.998) for DV1. The adsorption of the peptide was diminished by organic additives during the quantitative procedure. The intra- and inter-day precision was 1.9–9.8% and the accuracy was 91.2–110.0%. No significant variation was observed under the optimized conditions. The recovery was above 52% with low matrix effects. The method was successfully applied to a pharmacokinetic study of DV1 after subcutaneous injection at dose of 10 mg/kg in rats. The half-life and AUCinf of DV1 were calculated as 8.7 h and 35,553 ng/mL·h, respectively. It is the first report on the quantitative analysis and pharmacokinetic characterization of a D-peptide targeted CXCR4, which should be useful for further preclinical studies and development of this and other peptide therapeutics.