C-005 is a novel third-generation EGFR tyrosine kinase inhibitor for the treatment of non-small cell lung cancer (NSCLC). To support its clinical trial, we developed a rapid and sensitive bioanalytical method based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technique for the quantification of C-005 and its major metabolite in NSCLC patients following international bioanalytical guidelines. After a simple and quick protein precipitation step, the supernatant was injected to a Waters Acquity BEH C18 column (2.1 × 50 mm i.d., 1.7 mm), and the column was eluted with a gradient of buffer A (5 mM ammonium acetate and 0.1% formic acid in water) and buffer B (formic acid-acetonitrile (1:1000, v/v)). The eluates were subsequently detected by an AB QTRAP 5500 mass spectrometer with electrospray ionization using multiple-reaction monitoring mode. The method showed good linearity from 2.00 to 1000 ng/mL for C-005 and 1.00 to 500 ng/mL for M1. In conclusion, the validation results demonstrated the robustness of the method and its well-poised to support the first-in-patient study of C-005 in NSCLC patients. Copyright © 2020 Elsevier B.V. All rights reserved.