This new Methods in Enzymology chapter by Ben Smith, Jeff Gelles, and Bruce Goode “describe[s] techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described… complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks.”