Tyrosine phosphorylation regulates complex, multi-layered signaling networks with broad implications in (patho)physiology. To decipher these signaling networks directly in tissue samples we analyzed the EGF-dependent in-vivo phosphotyrosine signaling in lung tissue to a depth of more than one thousand phosphotyrosine sites. A phosphopeptide based proteomic screen identified proteins recruited to the EGF-regulated phosphotyrosine sites in lung tissue lysates. We demonstrate how cancer mutations near phosphotyrosine sites can introduce molecular switches that rewire signaling networks, and reveal how such a molecular switch is responsible for oncogenic properties of an EGFR lung cancer mutant. Introduction of cutting-edge LC-MS/MS instrumentation and data independent acquisition (DIA) enables scalable, rapid and high-throughput analysis of phosphotyrosine site interactions and phosphoproteomes. Our approach enables phospho-signaling investigations in tissues for in-depth mechanistic insights into oncogenic rewiring of signaling networks.