Formin homology proteins (formins) are responsible for the formation of actin structures such as actin stress fibers, actin cables, and cytokinetic contractile rings. Formins are the major actin filament (F-actin) nucleators in the cell. Because formins remain bound to the barbed end after nucleating an actin filament, it was expected that formins might rotate along the double-helical structure of F-actin during processive actin elongation (helical rotation). Here, we describe a method to detect the rotational movement of F-actin elongating from immobilized formins using single-molecule fluorescence polarization (FLP). Tetramethylrhodamine (TMR) attached to Cys-374 of actin emits polarized fluorescence at approximate to 45 degrees with respect to the filament axis. When the TMR-labeled F-actin laying at 45 degrees in the visual field rotates, the vertical- and horizontal-polarized fluorescence (FLV and FLH, respectively) of TMR alternately become bright. This technique allowed us to demonstrate the helical rotation of mDia1, a mammalian formin. Adenosine triphosphate (ATP) hydrolysis in actin subunits is not required for helical rotation; however, ATP appears to contribute to accelerating actin elongation by mDia1. When helical rotation is limited by trapping both mDia1 and the pointed-end side, the processive filament elongation is blocked. Thus, mDia1 faithfully rotates along the long-pitch helix of F-actin. In this chapter, we introduce the theoretical concept of single-molecule FLU, the optical setup, the preparation of adenosine diphosphatebound actin, and the procedure to observe the rotational movement of F-actin elongating from immobilized formins.