•The practical guidelines for wide-field microscopic imaging of actin filaments are demonstrated in both budding yeast and Arabidopsis.•Considerations for sample preparation and image acquisition are discussed.•The image processing and quantitative measurement approaches are provided for both yeast and plant studies.Eukaryotic cells depend on a dynamic actin cytoskeleton to regulate many conserved intracellular events such as endocytosis, morphogenesis, polarized cell growth, and cytokinesis (Engqvist-Goldstein and Drubin, 2003; Salbreux et al., 2012; Pruyne et al., 2004; Pollard, 2010). These activities depend on a precise and well-organized spatiotemporal actin assembly that involves many conserved processes found in eukaryotic cells ranging from a unicellular organism, such as yeast, to multicellular organisms, such as plants and human. In particular, both budding yeast Saccharomyces cerevisiae and plant Arabidopsis thaliana have been proven to be the powerful and great model organisms to study the molecular mechanisms of the polymerization of the actin cytoskeleton and the actin-driven processes in walled-cells. Here we describe the methods in imaging and image processing to analyze dynamic actin filament assembly in budding yeast and Arabidopsis using a wide-field fluorescent microscope.