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A selective, sensitive and high-throughput liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method has been developed and validated for the quantitation of Guanfacine in rat plasma. Sample clean-up involved liquid-liquid extraction (LLE) and 100 μL of rat plasma was used. YMC BASIC column (50 mm×2.0 mm, 3.5 µm) was used. Mobile phase used was 10 mM ammonium formate (pH 4.0):acetonitrile (70:30, v/v) at a flow rate of 0.3 mL/min. The parent→product ion transitions for the drug ( m / z 246.0→159.0) and IS ( m / z 252.0→161.1) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was validated over the concentration range of 50.00-10,000.00 pg/mL for Guanfacine. The method was successfully applied into a pharmacokinetic study in rat plasma.