A novel series of β-lactam derivatives that was designed and synthesized to target RGD-binding and leukocyte integrins is reported. The compound library was evaluated by investigating the effects on integrin-mediated cell adhesion and cell signaling in cell lines expressing αvβ3, αvβ5, αvβ6, α5β1, αIIbβ3, α4β1, and αLβ2 integrins. SAR analysis of the new series of azetidinones enabled the recognition of structural elements associated with integrin selectivity. We obtained selective and potent agonists that could induce cell adhesion and promote cell signaling mediated by αvβ3, αvβ5, α5β1, or α4β1 integrin, and antagonists for the integrins αvβ3 and α5β1, as well as α4β1 and αLβ2, preventing the effects elicited by the respective endogenous agonists.