A very large, ever-increasing repertoire of actin-binding proteins regulates the assembly dynamics and the spatial organization of actin filaments, thus orchestrating the motile behavior of the cell. The authors describe a series of biochemical functional assays that allow one to characterize the function of a putative actin-binding protein in actin filament dynamics. These tests allow the characterization of three types of actin-binding proteins: G-actin-sequestering proteins, profilin-like proteins, and barbed-end capping proteins. Biochemical tests include the use of sedimentation of actin filaments, polymerization assays at the barbed or pointed end of actin filaments derived from fluorescently labeled actin, thermodynamic measurements of actin assembly at steady state and during turnover of actin filaments, measurements of nucleotide exchange on G-actin, and the use of the intrinsic or extrinsic fluorescence of actin to measure direct binding of different protein ligands to G-actin.