Background: Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) having high efficacy in treating patients with advanced non-small cell lung cancer (NSCLC) with EGFR activating mutations and EGFR T70M mutations. Although, osimertinib is a frontline anticancer agent for treating NSCLC, many patients inevitably develop osimertinib-resistance tumor recurrence. The activation of AXL was reported to be a major factor driving osimertinib-resistance in NSCLC. Thus, AXL is a promising therapeutic target for the treatment of osimertinib-resistant NSCLC, and we developed a new small-molecule AXL inhibitor, AX-0085. Methods: We established osimertinib-resistant cell lines from EGFR-mutated NSCLC cell lines, HCC827 and H1975, after high dose exposure and stepwise exposure to osimertinib in culture. We investigated the cell viability of AX-0085 and other kinase inhibitors, and the anti-tumor activity of AX-0085 on the proliferation, migration, apoptosis, and epithelial-mesenchymal transition (EMT) in osimertinib-resistant cells. Results: Compared to the parental cells, IC₅₀ of resistant cell to osimertinib was significantly increased and osimertinib-resistant cells showed remarkably increased expression of AXL/pAXL and p-SRC as compared to the parental HCC827 cell. AX-0085 was completely blocked the AXL and SRC activation but EGFR inhibitors (gefitinib and osimertinib) did not inhibit the activation of AXL and SRC in the osimertinib-resistant cell line. AX-0085 (IC₅₀: 4.4nM for AXL kinase in cell-based kinase assay) was one of the most potent AXL inhibitors and showed the strongest cytotoxicity against osimertinib-resistant cells compared to other kinase inhibitors. AX-0085 effectively down-regulated mRNA level of AXL and AXL regulating transcription factors in NSCLC cell lines such as HCC827 and H1975. AX-0085 treatment effectively inhibited the activation of AXL in acquired osimertinib-resistant cells and decreased the mRNA level of AXL and AXL regulating transcription factors. AX-0085 significantly inhibited the cell proliferation, apoptosis, migration, and EMT in acquired osimertinib-resistant cells. Conclusions: Our results showed that AX-0085 selectively inhibits the activation of AXL to overcome acquired resistance to osimertinib, suggesting an effective AXL-based therapy for patients with osimertinib-resistant tumors. Currently, AX-0085 is undergoing non-clinical trials.