BACKGROUND The cytoskeleton consists of complex arrays of fibers that play indispensable roles in cell structure and function. The cytoskeletal fibers are concertedly involved in numerous cellular processes, including cell adhesion, locomotion, intracellular transport, and cell division. The organization of the cytoskeleton was extensively studied, mainly by immunofluorescence microscopy, yet these studies were mostly qualitative, and a reliable quantitative approach for determining fiber structure and distribution is still missing. METHODS In this study we developed algorithms for filament feature extraction, based on fluorescence microscopy. These algorithms are robust against blurring by slight defocus, high background, and noise, and are applicable to both fixed, immunolabeled cells and live cells expressing fluorescently tagged cytoskeletal proteins. The implemented FiberScore program is used in order to recognize, segment, and quantify various structural parameters of the cytoskeleton, including total fiber-associated fluorescence, as well as fiber length and orientation. Furthermore, these parameters can be determined for different cytoskeletal proteins in the same sample tagged with multiple-fluorescent labels, and the results can be correlated with other cellular parameters. RESULTS FiberScore was used here for the quantification of simultaneous changes in microtubule and actin filaments induced by the microtubule-disrupting drug nocodazole. Actin filaments, which are reported to respond reciprocally to microtubule disruption, are found to be affected by both immediate and delayed signals. CONCLUSIONS Analysis of the organization of fibers by the FiberScore algorithm allows quantification of the cytoskeletal signature of cells and offers reliable multiparametric functional assays for effects of drugs and other perturbations evaluated on a cell-by-cell basis.