In this study we used TKI sensitive (CALS) and resistant (CALR) human HNSCC cells grown as standard 2D monolayers, 3D spheroids or tumour xenografts alongside NMR spectroscopy to explore metabolic characteristics of acquired resistance to EGFR TKIs which, if independently validated, could have potential as minimally invasive biomarkers, and to assess the influence of the microenvironment on the metabolic readouts. Here, EGFR is linked to neoplasm.