The physiological function of the epidermal growth factor receptor (EGFR) is to regulate epithelial tissue development and homeostasis, but its overexpression and activation are strongly linked to oncogenesis and tumor progression in a variety of human cancers. Many EGFR-targeted therapies have been developed which alone have proven to be effective, but resistance often develops, indicating a need for more studies probing the efficacy of combinatorial drug treatments. These studies will further require the discovery and characterization of biomarkers that can predict therapeutic efficacy and resistance. The purpose of this study was to examine the effects of individual and combined treatment with two classical EGFR-targeted therapies with different modes of action on cellular models of human skin cancer and non-small cell lung carcinoma (NSCLC). We characterized the effects of a two-day treatment with varying concentrations of EGF on cellular health and proliferation and on the expression of several key biomarker proteins in A431 and A549 cell lines. We then examined the individual and combined effects of treatment with the tyrosine kinase inhibitor (TKI) gefitinib and the humanized monoclonal antibody cetuximab. Cellular growth, proliferation and health were assessed with ATPlite luminescence assays and well-imaging and cytometry measures using a high-throughput multimodal imager and plate reader. Biomarkers measured included EGFR, immune checkpoint proteins (PD-L1 and CD276/B7-H3), chemokines (IL-8/CXCL8 and GRO-α/CXCL1), a protease inhibitor (TIMP-2), and growth factor (VEGF). All these biomarker proteins were rapidly assessed using AlphaLISA no-wash, mix-and-read assays on 5 μL samples collected from the same wells of the culture plate. We observed that EGF treatment resulted in decreased EGFR expression and upregulation of PD-L1 protein in a dose-dependent manner in both culture models. CD276, VEGF and TIMP-2 expression levels, however, were upregulated with EGF treatment only in the A431 cells. Complex drug treatment effects on biomarker expression are presented. In addition, treatment effects on EGFR phosphorylation and downstream signaling pathways (including MEK/ERK and AKT) were probed using AlphaLISA SureFire Ultra phosphorylation assays and demonstrate the different pathways effected by either drug or a combination of both. These data illustrate the effects of EGFR stimulation on the expression of an array of biomarkers and signaling pathways and in the cellular tolerance for individual and combined treatment with two EGFR-targeted therapies with different mechanisms of action between two different cellular models of human cancer. Citation Format: Jeanine M. Hinterneder, Lauren Berstler, Adam Carlson, Jen Carlstrom. A systems approach to evaluating the effects of two EGFR-targeted therapies on cellular models of human skin and lung cancer using an integrated imaging and analysis platform and the rapid quantification of multiple key biomarkers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5197.