Increased frequency of CD4+CD25+regulatory T-cells (Treg) has been associated with disease progression in chronic lymphocytic leukemia (CLL). Flow cytometric methods, which allow for the simultaneous analysis of their specific transcription factor Foxp3 and activated STAT proteins, together with proliferation can help to elucidate the signaling mechanisms driving Treg expansion and suppression of FOXP3- conventional CD4+T-cells (Tcon). Herein, we first report a novel approach in which STAT5 phosphorylation (pSTAT5) and proliferation (BrdU-FITC incorporation) could be analyzed specifically in FOXP3+ and FOXP3- responding cells after CD3/CD28 stimulation. The addition of magnetically purified CD4+CD25+T-cells from healthy donors to cocultured autologous CD4+CD25−T-cells resulted in suppression of Tcon cell cycle progression accompanied by a decrease in pSTAT5. Next, a method using imaging flow cytometry is presented for the detection of cytokine-dependent pSTAT5 nuclear translocation in FOXP3-expressing cells. Finally, we discuss our experimental data obtained by combining Treg pSTAT5 analysis and antigen-specific stimulation with SARS-CoV-2 antigens. Applying these methods on samples from patients revealed Treg responses to antigen-specific stimulation and significantly higher basal pSTAT5 in CLL patients treated with immunochemotherapy. Thus, we speculate that through the use of this pharmacodynamic tool, the efficacy of immunosuppressive drugs and their possible off-target effects can be assessed.