Here we used kinase affinity enrichment (Kinobeads) and intensity-based mass spectrometry to elucidate aquired resistance mechanisms to EGFR inhibition in cancer cells. First, kinase expression differences between untreated control cells and long term treated resistant cells were investigated using Kinobeads and LC-MS/MS. Overexpressed Kinases were then further investigated by cellular assays to evaluate targets and development of combination treatment strategies.