The recent clinical success of cancer immunotherapy with checkpoint blockade has led to renewed interest into the development of immune modulatory agents with the capacity to?activate?anti-tumor?T cell?responses. Standardization of optimized?in vitro?assays?for efficient assessment of immune?function?of such new drugs is thus needed to facilitate clinical development of the optimal drug candidates. Here, we describe an optimized version of?T cell?suppression?assay?designed to test the effect of?immunomodulatory?agents on?T cell?function?and?activation. We apply this?assay?to investigate the agonist?activity?of the?T cell?co-stimulatory molecule glucocorticoid-induced TNFR-related protein (GITR). We detail a protocol for concurrent assessment of multiple levels of?T cell?functional?modulation upon GITR engagement, including?T cell?priming,?activation?and?effector?function, in a single?assay. As human GITR agonist?antibodies?are currently under development, availability of standardized cell-based?functional?assays?of GITR agonism is instrumental to translate anti-GITR therapy into the clinical setting.