Key considerations when selecting an appropriate Pharmacodynamic (PD) Biomarker are accessibility of material and sensitivity of detection and quantification. The answers to these questions will dictate the design and validation of an appropriate detection method. This can be exemplified in two settings of colorectal cancer (CRC) and multiple sclerosis (MS); where regulatory T Cells (Tregs) are believed to play important roles in disease progression. While FoxP3 staining in tissue biopsies is a useful tool to determine Treg distribution and quantification, these invasive procedures are usually restricted for CRC and not an option for MS. However, circulating Treg levels have been reported to increase (CRC) or decrease (MS) according to disease progression. An accurate and robust flow cytometry panel can document altered cell frequencies in peripheral blood. This allows monitoring of disease progression in patients and their response to treatment. We describe a validated and robust flow cytometry panel to precisely monitor changes in Treg frequencies as a PD biomarker to complement immunohistochemistry methods.