Updated project metadata. Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase is overexpressed in 90% of Head and neck squamous cell carcinoma (HNSCC) patients. Clinical trials with EGFR-targeted tyrosine kinase inhibitors such as erlotinib have shown a modest activity in HNSCC alternate mechanisms of resistance are acquired. To investigate these acquired mechanisms of resistance and identify novel therapeutic targets we employed SILAC-based tyrosine phosphoteomic analysis of an isogenic pair of erlotinib sensitive (SCC-S) and resistant (SCC-R) HNSCC cell line. Quantitative phosphotyrosine profiling revealed 98 phosphopeptides belonging to 64 proteins and 66 phosphopeptides belonging to 52 proteins to be hyper and hypophosphorylated (≥2 fold) in SCC-R cells, respectively. Several proteins such MET proto-oncogene, receptor tyrosine kinase (MET) and CRK like proto-oncogene, adaptor protein (CRKL) known to mediate erlotinib resistance in HNSCC and lung cancer were found to be dysregulated. Bioinformatics analysis of differentially phosphorylated proteins showed enrichment of proteins involved in focal adhesion kinase (FAK) pathway downstream of EGFR. We identified and validated activation of several phosphorylation sites of protein tyrosine kinase 2 (PTK2) in SCC-R cells and its downstream targets. We further demonstrated that CUB-domain containing protein 1 (CDCP1), an upstream regulator of PTK2 activity and PTK2 can be targeted in combination as an alternative to erlotinib in HNSCC.