Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase which is regarded as a critical signalling component involved in tumour progression and metastasis. It is an important mediator in cell proliferation and survival, hence, an imperative therapeutic target in breast cancer treatment. The aim of this study was to determine the effect that extracellular matrix (ECM) has on FAK expression and efficacy of known FAK inhibitors, when used against breast cancer cells. Breast cancer cell lines, MCF-7, BT-474 and MDA-MB-231 were grown in the absence and presence of Matrigel and the levels of FAK expression were measured by immunofluorescence. Cells grown in the presence and absence of Matrigel were treated with FAK inhibitors; Y15 and TAE226. The expression levels of FAK and the IC50 values of the inhibitors determined. The results indicated that in all breast cancer cell lines tested, FAK expression is elevated when grown on Matrigel in limited amounts enabling monolayer growth. All cell lines tested exhibited more sensitivity to FAK inhibitors in the presence of Matrigel resulting in up to 50% reduction in IC50 values compared to those obtained in the absence of Matrigel. These results suggest a role for ECM proteins in the efficacy of FAK inhibition with Y15 and TAE226, with TAE226 having greater effect as anticipated.