Regulatory T cells (Tregs) play an important role in regulating immune homeostasis in health and disease. Traditionally their suppressive function has been assayed by mixing purified cell populations, which does not provide an accurate picture of a physiologically relevant immune response. To overcome this limitation, we here developed “single cell suppression profiling of human Tregs” (scSPOT). scSPOT is more informative and physiological than traditional assays, using a 52-marker CyTOF panel, a cell division detection algorithm, and a whole PBMC system to assess the function of Tregs on all cell types simultaneously. By employing scSPOT, we observed Tregs having the clearest suppressive effects on CD8-EM through division arrest, cell cycle inhibition, and effector molecule downregulation. Additionally, we demonstrate cellular differentiation tracking and drug screening applications of scSPOT and use it to identify the potential origin of a Treg phenotypic split in critical viral infection and further propose modes of action by the FDA-approved drugs Ipilimumab and Tazemetostat. scSPOT is scalable, robust, widely applicable, and may be used to better understand Treg immunobiology and screen for therapeutic compounds.