Abstract Although small molecule inhibitors of BRAF have shown great promise in advanced melanoma, resistance is commonplace. Many patients with acquired BRAF and BRAF/MEK inhibitor resistance develop disease at new sites, suggesting that drug-induced selection pressure drives metastasis. Here we used mass spectrometry-based phosphoproteomic screening and network modeling to uncover two novel adaptations to BRAF inhibitor therapy. The first involved ligand-independent EphA2 signaling that led to the adoption of an invasive/metastatic phenotype. The EphA2-mediated invasion was epigenetically mediated, AKT-dependent and was readily reversible upon removal of drug as well as through PI3K or HDAC inhibition. In a human melanoma mouse xenograft model, chronic BRAF inhibition led to the development of metastases, which stained positively for EphA2. A second phosphoproteomic screen showed BRAF inhibition to enhance the survival of both melanoma cells and fibroblasts through the induction of fibronectin (FN)/integrin α5β1 signaling. Adhesion to FN amplified the adaptive EGFR, HER2 and c-MET signals required for PI3K/AKT-mediated survival when BRAF was inhibited. At the same time, BRAF inhibition led, directly and indirectly, to the paracrine release of HGF and neuregulin from fibroblasts, with TGF-β release from the melanoma cells increasing both fibroblast differentiation and survival. These effects were partly mediated through paradoxical MAPK activation in the fibroblasts and could be reversed through BRAF/MEK inhibition. Thus, we have used comprehensive phosphoproteomics to identify two key non-genetic adaptations to BRAF inhibitor therapy that offer new insights into the process of therapeutic escape. Citation Format: Inna V. Fedorenko, Kim H.T. Paraiso, Bin Fang, John M. Koomen, Keiran S.M. Smalley. Using comprehensive proteomic approaches to map signaling adaptations to BRAF and BRAF/MEK inhibition. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr IA04.