In order to study how dynamic changes of α5β1 integrin engagement affect cellular behaviour, photoactivatable derivatives of α5β1 specific ligands are presented in this article. The presence of the photoremovable protecting group (PRPG) introduced at a relevant position for integrin recognition, temporally inhibits ligand bioactivity. Light exposure at cell-compatible dose efficiently cleaves the PRPG and restores functionality. Selective cell response (attachment, spreading, migration) to the activated ligand on the surface is achieved upon controlled exposure. Spatial and temporal control of the cellular response is demonstrated, including the possibility to in situ activation. Photoactivatable integrin-selective ligands in model microenvironments will allow the study of cellular behavior in response to changes in the activation of individual integrins as consequence of dynamic variations of matrix composition.