This project highlighted that Src and Src kinase family (SFK) members play a definitive role in breast cancer. Due to the paucity of translational studies, we investigated if SFK members are expressed in human breast tissue. Eight SFK members were present with distinct mRNA expression patterns in normal, non-malignant and malignant breast tissue. Immunohistochemistry was employed to investigate protein expression and activation of Src and SFK members. Survival analysis revealed that c-Src and activated Y419Src were associated with worse patient outcome, confirming current in vitro literature, whereas a different phosphorylation site of Src (Y215) and expression of Lck was associated with improved clinical outcome. Dasatinib was employed in different breast cancer cell lines to establish its effect on those phosphorylation sites. Decreased expression of c-Src and Y419Src was observed, whilst Y215Src expression stayed unchanged, providing a rationale for using this Src kinase inhibitor in clinical trials.