3′,5′-Cyclic adenosine monophosphate (cAMP) is a second messenger with diverse actions and strong immunomodulatory effects. For example, in T cells, cAMP regulates a myriad of functional responses ranging from peripheral maintenance of naïve T cells and their activation via T cell receptors to the execution of effector functions. cAMP level in the cell is regulated by adenylyl cyclases (ACs) and phosphodiesterases (PDEs). While ACs get activated after stimulation of G-protein coupled receptors on the surface of CD4+ T cells, PDEs hydrolyze cAMP and thus ensure the balance between its synthesis and degradation. High cAMP levels in regulatory T cells (Treg) as compared to conventional T cells are known to be one of the central components of Treg-mediated immunosuppression. To monitor cAMP levels at a single-cell level, Förster resonance energy transfer (FRET) is employed for live cell imaging. Here, we describe how to perform real-time FRET measurements in activated CD4+ T cell subsets isolated from transgenic mice ubiquitously expressing Epac1-camps biosensor. In these cells, cAMP levels can be increased by adenosine A2A receptor agonist or by inhibiting PDE activity with the pan-PDE inhibitor IBMX. This protocol can be generally applied to various T cell populations under different conditions.