Quantifying the mechanics of the cytoskeletons of living cells is important for understanding several physiologic and pathologic cellular functions, such as wound healing and cellular migration in cancer. Our laboratory develops three-dimensional tissue constructs for assaying cytoskeletal mechanics in controlled conditions. These tissue constructs consist of defined components such as chick embryo fibroblasts and reconstituted rat tail collagen; fibroblasts remodel the collagen extracellular matrix (ECM) and develop a structural environment representative of that which would exist in a natural tissue. Our protocol for quantifying the microscale mechanics of the proteins that comprise the cytoskeleton involves mechanical testing of a tissue construct first in a bath that contains nutrition medium to support the active physiologic functioning of the cells, and next in the presence of inhibitors that selectively eliminate specific cytoskeletal structures. By solving an inverse homogenization problem, the mechanical functioning of these proteins at the cellular level can be estimated. Here, we present a combination of mechanical testing and imaging results to quantify the effects of specific inhibitors on cytoskeletal and extracellular matrix form and function.