Integrins are key signalling molecules that mediate the complex relationship between cancer cells and their associated extracellular matrix. As such, they have been implicated in every step of cancer progression. Their cytoplasmic domains are essential hubs for protein-protein interactions that are often modulated through phosphorylation of key residues. Despite the established importance of these residues, the regulators of integrin phosphorylation remain elusive. To address this, we have developed and optimised a Förster resonance energy transfer (FRET) biosensor for integrin β1 phosphorylation, which we have applied to screen for regulatory phosphatases from the 107 known protein tyrosine phosphatases (PTPs) in the human genome. In parallel, we have generated cell lines with RFP-tagged integrin β1 where the NPxY(783/795) sites have been mutated to phenylalanines. Using these cell lines, we found that loss of the integrin phosphorylation dynamics in breast cancer cells dramatically reduced the formation of invadopodia on fluorescent gelatin, as well as invasion into three-dimensional organotypic co-cultures, which was again observed when inhibiting the regulatory PTP, Shp2, identified in the FRET screen. In order to elucidate the molecular pathways that form the phosphorylated integrin complex, we employed the adaptor protein Dok1 (the only known interactor of phosphorylated integrin β1) and applied bimolecular complementation assays to immunoprecipitate the integrin β1/Dok1 complex for mass spectrometry. This allowed us to identify novel interactors, including VPS35, Cofilin and Annexin A6, that we are now assessing for changes in their recruitment to invadopodia upon inhibition of PTPs or integrin kinases, such as Arg and Src (validated in this study). Along with mechanistic assessment, future work will involve interrogation of the clinical relevance of targeting components of the phosphorylated integrin complex, which will then be evaluated with immunohistochemistry staining of large breast cancer cohorts, along with functional validation using patient material from clinical collaborators. These aspects will provide an essential link from the mechanistic insights to improving patient outcomes from this work.