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Background Acquired resistance to tyrosine kinase inhibitors (TKIs) is becoming a major challenge in the treatment of many cancers. Epidermal growth factor receptor (EGFR) is overexpressed in squamous carcinomas, notably those of the head and neck (HNSCC), and can be targeted with several TKIs. We aimed to identify soluble proteins suitable for development as markers of EGFR TKI resistance in cancer patients to aid in early and minimally invasive assessment of therapeutic responses. Methods Resistant HNSCC cell lines were generated by exposure to an EGFR TKI, gefitinib, in vitro . Cell lines were characterised for their biological behaviour in vitro (using growth inhibition assays, flow cytometry, western blots, antibody arrays and/or immunoassays) and in vivo (using subcutaneous tumour xenografts). Sera from EGFR-treated and -untreated HNSCC patients were analysed by immunoassay. Results Two independent sublines of CAL 27 and a PJ34 subline with acquired resistance to EGFR TKIs (gefitinib, erlotinib and afatinib) were developed. Resistant cells grew as highly aggressive xenografts leading to reduced host survival rates compared with EGFR-TKI sensitive cells. This suggested a link between resistance in vitro and poor prognosis in vivo . A significant upregulation of proteins linked to tumour angiogenesis and invasion was identified in resistant cells. This ‘resistance-associated protein signature' (RAPS) was detected in the sera of a small cohort of HNSCC patients and was associated with reduced survival. Conclusion We have identified a protein signature associated with EGFR-TKI resistance that may also be linked to poor prognosis and warrants further investigation as a potential clinical biomarker.