Abstract <p><b>Objective</b>: The cytoskeleton is a complex dynamic cellular structure that not only serves as a scaffold but also participates in cell motility, metabolism, and regulation of biochemical processes. Studying the reaction of the cytoskeleton to various stimuli is an important task of cell biology. Automated microscopy and image processing methods are currently being developed to obtain and analyze extensive volumes of objective data. The aim of this study was to develop an approach for the objective quantitative analysis of cytoskeletal components using CellProfiler and ImageJ software. <b>Methods:</b> CellProfiler, ImageJ, and the Cellpose algorithm were used to segment and analyze fluorescence microphotographs of actin filaments, microtubules, and intermediate filaments. <b>Results:</b> An approach for high-throughput image processing with the ability to analyze the shape, intensity, and fractal dimensions of the cytoskeleton was developed. <b>Conclusion:</b> The proposed approach to cytoskeleton analysis enables high-throughput analysis of a large number of cell images, thereby obtaining a vast amount of diverse data on fluorescence internsity and cytoskeletal structure.</p>