Two categories of regulatory T cells (Tregs), nTreg and iTreg, play vital roles in orchestrating the integrity of a host in the course of an immune response. Tregs commonly belong to CD4+ CD25+ T cells and they are characterized by a transcription factor – forkhead box P3 (FoxP3). Within the space of the last few years, interests have been drawn to Tregs as a therapeutic tool in several settings like autoimmune disease, transplantation, and tumor disorders. As a consequence, to assess the functional properties of Tregs, namely through their ability to suppress other cells, cytokine expression, and proliferation in a variety of conditions, it is mandatory to gain better approaches to this end. This would be beneficial in designing better-than-ever therapeutic methods with regard to Tregs properties. Gaining better insights into the underlying mechanisms of immune regulation, by means of straightforward and less time-consuming techniques, will hopefully permit the therapeutic application of these cells in the control of human disorders. This review aims at going through the basic methods for Treg isolation as well the efficiency of the commonly exerted in vitro assays of Tregs-mediated immune suppression.