In autoimmune diseases or transplant graft rejection, a therapy that will prevent or reduce the present immune activation is highly desired. Ex vivo generated tolerogenic dendritic cells (DC) are considered to have a strong potential as cellular therapy for these diseases. One of the mechanisms of immune suppression mediated by tolerogenic DC is the induction of regulatory T-cells (Treg). Consequently, the efficacy of such DC to induce Treg will reflect their tolerogenic capacity. Because no specific markers have been described for human induced (i)Treg yet, the Treg can only be appreciated by functionality. Therefore, we have optimized an in vitro suppression assay to screen for human DC-induced-Treg activity. IL-10-generated tolerogenic DC were used to induce Treg that were previously shown to effectively suppress the proliferation of responder T-cells stimulated with allogeneic mature DC (mDC). Our results show that the suppressive capacity of IL-10 DC-induced Treg measured in the suppression assay increases with the iTreg dose and decreases with higher numbers of antigen-presenting cells (APC) as T-cell stimulation. Lowering the ratio between responder T-cells and stimulator mDC present in the coculture clearly improved the read-out of the suppression assay. Furthermore, mDC-primed T-cells in the suppression assay were shown to be an essential control condition. In conclusion, we recommend titrations of both APC and iTreg in the suppression assay and to include a negative control condition with T-cells primed by mDC, to distinguish specific and functional suppression by iTreg from possible generalized suppressive activity.