FOXP3(+) regulatory T cells (Tregs) play an important role in controlling immune activation, maintenance of -homeostasis and the prevention of autoimmunity. Much effort has been focused in assessing their potential defects in certain human diseases and in developing potential Treg immunotherapy to cure autoimmune conditions. One cardinal feature of Tregs is their ability to suppress the activation and proliferation of effector T cells. The in vitro suppression assay is a convenient and relatively reliable method to quantify their suppressive function and validate their identity as true Tregs. This protocol describes a reliable, nonradioactive in vitro suppression assay to assess the immunosuppressive property of human Tregs in inhibiting proliferation of dye-labeled responder T cells using non-irradiated, HLA-DR(+) antigen presenting cells and soluble anti-CD3 antibodies as stimuli.