sActin cytoskeleton plays a critical role in regulating T cell motility and activation. However, the lack of a realtime quantitative method to analyze actin assembly has limited the progress toward understanding actin regulation. Here, we describe a novel approach to probe actin dynamics on living T cells using FRET combined with flow cytometry. We have first generated a Jurkat T cell line stably coexpressing EGFP and mOrange FPs fused to actin. The real-time variation of actin monomer assembly or disassembly into filaments was quantified using a ratiometric flow cytometry method measuring changes in the mOrange/EGFP emission ratio. The method was validated on resting T cells by using chemical compounds with known effects on actin filaments and comparison with conventional microscopy imaging. Our method also detected the rapid and transient actin assembly in T cells stimulated by anti-CD3/CD28- coated beads, demonstrating its robustness and high sensitivity. Finally, we provide evidence that lentiviral-mediated transduction of shRNAs in engineered Jurkat cells could be used as a strategy to identify regulators of actin remodeling. In conclusion, the flow cytometric FRET analysis of actin polymerization represents a new technical advance to study the dynamics of actin regulation in intact cells.