Little is known about lung carcinoma EGF kinase pathway signaling within the context of the tissue microenvironment. We quantitatively profiled the phosphorylation and abundance of signal pathway proteins relevant to the EGF receptor within Laser Capture Microdissected (LCM) untreated, human non-small cell carcinomas (NSCLC) (n=25) of known EGFR tyrosine kinase domain mutation status. We measured six phosphorylation sites on EGFR to evaluate whether EGFR mutation status in vivo was associated with the coordinated phosphorylation of specific multiple phosphorylation sites on the EGFR and downstream proteins. Reverse phase protein array quantitation of NSCLC revealed simultaneous increased phosphorylation of EGFR residues Y1148(p<0.044) and Y1068(p<0.026), and decreased phosphorlation of EGFR Y1045(p<0.002), Her2 Y1248(p<0.015), IRS-1 S612(p<0.001), and Smad S465/467(p<0.011), across all classes of mutated EGFR patient samples compared to wild type. To explore which subset of correlations was influenced by ligand induction versus an intrinsic phenotype of the EGFR mutants, we profiled the time course of 115 cellular signal proteins for EGF ligand stimulated (3 dosages) NSCLC mutant and wild type cultured cell lines. EGFR mutant cell lines (H1975 L858R) displayed a similar pattern of EGFR Y1045 and Her2 Y1248 phosphorylation to that found in tissue. Persistence of phosphorylation for AKT S473, following ligand stimulation, was found for the mutant. These data suggest that a higher proportion of the EGFR mutant carcinoma cells may exhibit activation of the PI3K/ AKT /mTOR pathway through Y1148 and Y1068 and suppression of IRS-1 S612, altered heterodimerization with ERBB2, reduced response to TGFß suppression, and reduced ubiquitination/degradation of the EGFR through EGFR Y1045, thus providing a survival advantage. This is the first comparison of multiple, site-specific phosphoproteins to the EGFR tyrosine kinase domain mutation status in vivo.