Metastasis is the leading cause of death among breast cancer patients, and drugs targeting metastatic processes are much needed. Screening for compounds to inhibit such processes in an assay format reminiscent of in vivo conditions should increase the likelihood of finding suitable compounds. We have established a three-dimensional in vitro cellular assay to simulate conditions of disseminated tumour cells, and have used it to screen a library of kinase inhibitors for compounds that inhibit growth of metastatic human breast cancer cells. As a reference, we have also screened the same compounds in a standard two-dimensional assay. From our screen, a set of compounds was found that had an increased inhibitory effect on cells growing in a three-dimensional matrix compared to cells growing directly on a plastic surface. Of particular interest was a set of compounds targeting focal adhesion kinase (FAK), Src and PI-3K/mTOR, showing significantly increased potency in the three-dimensional assay compared with normal tissue culture conditions. Contact between cells and extracellular matrix is mediated by cell surface integrins to the intracellular signalling networks through adapter proteins such as Src and FAK. This signalling can affect many other pathways, e.g. pathways activated by receptor tyrosine kinases, and can influence cellular processes relevant to metastasis such as growth, invasiveness and motility. Thus, inhibition of this pathway, either on its own or in combination with other pathways, could be an attractive strategy to target metastatic disease.