Osimertinib (AZD9291) is a highly selective irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) designed to treat nonsmall-cell lung cancer patients with EGFR active and T790M resistant mutations. A rapid and sensitive method for quantitative analysis by ultra-performance liquid chromatography-tandem mass spectrometry of osimertinib and its metabolite AZ5104 in human plasma was developed and validated. The samples were prepared by protein precipitation and separated on a BEH C-18 column (2.1 x 50 mm, 1.7 mu m) by gradient elution with 0.1% (v/v) formic acid and 10 mM ammonium acetate in water and acetonitrile as the mobile phase. Electrospray ionization in positive ion mode and multiple reaction monitoring were used to monitor the ion transitions at m/z 500.4 -> 385.3 and 486.3 -> 371.1. The results indicated that the method had excellent sensitivity and specificity. The validation was performed in a range from 0.5 to 100 ng/mL. Intraday and inter-day precisions (in terms of RSD) were all <15%, and the accuracies (in terms of RE) were within +/- 15%. The lower limit of quantification, matrix effect, extraction recovery, stability and dilution integrity were also validated and satisfied the validation criteria. Finally, this method was successfully applied in a retrospective analysis, and the predose samples of 52 nonsmall-cell lung cancer patients who were enrolled in a novel third EGFR TKI clinical trial were analyzed for screening regardless of whether they had previously received osimertinib treatments.