Oncogene-targeted therapies against growth factor receptors are effective treatment options for driver mutation-positive non-small cell lung cancer (NSCLC). The third-generation EGFR-Tyrosine Kinase inhibitor (TKI) osimertinib, is a standard first-line therapy for patients with EGFR-mutated NSCLC cancer. Acquired resistance to osimertinib is a significant problem that limits survival. In addition to that clinical data show approximately 15% of EGFR mutant non-small cell lung cancer patients have innate resistance to Osimertinib. Tumor heterogeneity and multiple resistance mechanisms add to the complexity of EGFR-mutated NSCLC. Tumor cells that acquire resistance, independent of the mechanism, experience replication stress (RS) as proliferation resumes. Methods: We have employed a series of non-small cell lung cancer models to assess the efficacy of combining osimertinib with NERx-329, targeting RS via inhibiting RPA, and the impact of these treatments on growth and damage signaling pathways. Results: We demonstrate that targeting RS with RPAi treatment induces cell death in multiple EGFR-mutant cell lines alone and when combined with osimer-tinib. Dissection of signaling pathways revealed that RPAi treatment does not block osimertinib inhibition of EGFR signaling. Analysis of DNA damage response (DDR) signaling reveals that NERx-329 potentiates the osimertinib-dependent downregulation of Chk1 activity and expression. The Chk1 loss was shown to be dependent on proteasome degradation as proteasome inhibitor MG-132 restores Chk1 activity and expression. Conclusions: From these data, we infer that targeting RS via RPAi NERx-329 in combination with osimertinib is an effective strategy and represents a promising drug combination targeted therapy to enhance efficacy and limit the development of resistance.