The Src family kinases (SFKs) are comprised of nine highly homologous members that participate in various signaling pathways, to regulate events such as proliferation, differentiation, cell migration, adhesion, and survival. While Src has been intensively studied, less is known about other family members. Unfortunately, similarities in the structure and regulation of SFKs have made it difficult to dissect their unique functions. Differential expression of individual SFKs have been found in various cancer cell lines, yet their functional specificity remains largely unknown. Previous strategies to investigate SFK specificity, such as overexpression and use of non-selective inhibitors, have been hampered by genetic compensation or lack of specificity. Therefore, it was imperative that new tools be developed in order to identify specific roles for individual SFKs. This dissertation describes two strategies to manipulate the activity of SFKs and to reveal novel information about their functional specificity in cell motility. The first study I performed used a small molecule to specifically activate each SFK isoform and to elucidate possible mechanisms contributing to the distinct activation phenotypes of Src and a Src family member, Fyn. In this study, new computational methods were developed to automatically analyze the dynamics of morphological changes. In the second study, I used light to manipulate Src activity. These strategies not only provide absolute control of specific Src family members, but also allow for spatial control of kinase activity. Additionally, these strategies utilize the same conserved site in the catalytic domain of the kinase and can be broadly applied to other tyrosine kinases.