Regulatory T cells (Tregs) are a rare subset of lymphocytes that inhibit the activation and effector functions of T cells and are important regulators of immune responses. Although Tregs are well characterized in humans and rodents, little is known about their immunophenotyping (IP) profile in cynomolgus macaques (Macaca fascicularis), which is an important species for pharmacological and toxicological evaluation of potential immune modulators because of their similar physiologic, genetic, and metabolic response patterns to humans. The authors have developed an immunophenotyping panel using a high-throughput 96-well microtiter plate-based assay to detect circulating Tregs (CD3+CD4+CD25hiFoxP3+) and have determined the normal range for the number of Tregs in naive healthy cynomolgus macaques to be 56.4 to 179.7 cells/μL (mean ± SEM = 113.6± 5.1 cells/μL; n = 25). Furthermore, the authors compared the resulting FoxP3+ Treg profiles with a CD127lo cell-surface panel (CD3+CD4+CD25hi CD127lo) and found a close correlation between the absolute numbers of CD3+CD4+CD25hiFoxP3+ and CD3+CD4+CD25hiCD127lo cells (mean ± SD = 120 ± 8.0 cells/μL). Quantification of circulating Tregs in cynomolgus macaques in this high-throughput assay may help to identify drug candidates that affect this rare, but critical, immunoregulatory cell population.