Fibronectin (Fn) is an adhesive extracellular matrix protein assembled by fibroblasts into fibrils within ECMs of developing and remodeling tissues. Fn is sensitive to mechanical forces exerted by contractile cells, and can alter its structural conformations in response to mechanical strain within Fn fibrils. We developed probes (both peptide and antibody) to Fn to detect mechano-sensitive perturbations of Fn conformation. Probes were characterized for their binding characteristics (affinity, epitope, mechano-sensitivity) and validated on multiple in vitro and in vivo ECM models. Furthermore, we showed that the mechano-sensitive H5 antibody that we have developed have utility in detection of early molecular signatures of fibrosis in vivo in a mouse model of pulmonary fibrosis. Using the H5 antibody, we also report detection of a conformational switch within the integrin binding FnIII9-10 region. Modulation of Fn’s integrin switching behavior may help in the development of controllable “smart” biomaterials, as well as to the development of conformation-specific imaging probes to detect early molecular signatures of tumor and fibrotic ECMs.