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A simple and sensitive high performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) method for determination of AZD3759, a novel epidermal growth factor receptor tyrosine kinase inhibitor, in rat plasma and brain homogenate was developed and validated over the range of 1.0–1000 ng/mL. Chromatographic separation was carried out on a C18 column with acetonitrile and 0.1% formic acid in water as mobile phase with gradient elution at a flow rate of 0.4 mL/min. The lower limits of quantification (LLOQs) were 1.0 ng/mL for AZD3759 in both rat plasma and brain homogenate. The intra-day and inter-day precision and accuracy of AZD3759 were well within the acceptable limits of variation. The simple and sensitive LC–MS/MS method was successfully applied to the pharmacokinetic and brain distribution studies following an oral administration of AZD3759 to rats.The concentration of AZD3759 in rat biomatrices was determined by LC–MS/MS.The pharmacokinetic and brain distribution of AZD3759 in rats were investigated.The results would be useful in further applications of AZD3759.