Lipid membranes provide physical and biochemical boundaries that compartmentalize intracellular functions and create the modular structure of the cell. The different membranous compartments within eukaryotic cells, such as the Golgi apparatus, the endoplasmic reticulum and the nucleus, although separate, are in communication with each other. These compartments are shaped, remodeled, and connected by their continual interaction with the mechanically active and force generating components of the cell, cytoskeletal filaments and cytoskeletal motors. The interplay among lipid membranes, the cytoskeleton, and motors establishes a crosstalk between mechanical forces and biochemical signaling to control many aspects of the cell including its morphology, its internal architecture, vesicular cargo trafficking, and gene expression. The need for a more mechanistic understanding of how lipid membranes, cytoskeletal filaments and motors coordinate to generate and transduce mechanical forces has led to the development of in vitro reconstitution assays of independently functioning modules involving the minimal components. Technological advances in biophysical research over the last two decades have allowed researchers to build up assays of increased complexity which has allowed validation of models based on in vivo observations, identification of new emergent properties, and new predictions for more targeted in vivo research. The goal of this collection is to present detailed protocols for the study of the interaction between lipid and cytoskeletal components that can further inspire biophysicists and cell biologists to expand upon them and continue to develop the field.