High incidence and mortality rates for non-small-cell lung cancer (NSCLC) lead to low survival rates. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) are commonly first prescribed for NSCLC patients with EGFR mutations. However, most patients with sensitizing EGFR mutations become resistant to EGFR-TKI after 9-13 months treatment. Yiqi Chutan Tang (YQCT) has been prescribed as a treatment to this issue for over 20 years. In this report, high-performance liquid chromatography (HPLC) analysis, flow cytometry, western blot analysis, and functional annotation analysis were applied to uncover the molecular mechanisms of YQCT. Our results show the application of YQCT reduces gefitinib-induced drug resistance, induces slight cell cycle arrest, enhances gefitinib-induced apoptosis, and activates the autophagy. These results indicate that at the molecular level YQCT can reduce drug resistance and improve anti-cancer effects when associated with gefitinib, which could be a result of enhancement of apoptosis and autophagy in the EGFR-TKI resistant cells of NSCLC. This research provides a new treatment strategy for patients with EGFR-TKI resistance in NSCLC. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.