Regulatory T cells (Treg) are a subset of T lymphocytes that in humans represent less than the 10 % of circulating CD4(+) T cells. Treg are specialized in the inhibition of the immune responses and play a crucial role in the maintenance of immunological tolerance. Several lines of evidence clearly documented the role of Treg in restraining antitumor immune responses. For this reason, antitumor immunotherapy approaches have been recently associated with drug treatments aimed at depleting Treg or blocking their functions. A summary of the currently used in vivo approaches to limit Treg expansion in cancer patients is here provided.A comprehensive phenotypic and functional monitoring of Treg is crucial for the precise assessment of the effects that these different drug treatments exert on Treg. In this chapter, we will provide guidelines for an accurate ex vivo identification of human Treg. Due to the phenotypic and functional heterogeneity, intrinsic plasticity, and the lack of a unique marker exclusively expressed by human Treg, the clear-cut identification of this T cell subset requires the expert usage of multiparametric flow cytometry analysis (FACS). In this view, a combination of phenotypic and functional assessment of Treg is mandatory. In this chapter, we will describe the most reliable methods to identify and monitor the modulation of human Treg in patients undergoing immunological or drug-based treatments. Protocols to measure ex vivo the suppressive functions of Treg are also provided.