Applied forces and the biophysical nature of the cellular microenvironment play a central role in determining cellular behavior. Specifically, forces due to cell contraction are transmitted into structural ECM proteins and these forces are presumed to activate integrin “switches.” The mechanism of such switches is thought to be the partial unfolding of integrin-binding domains within fibronectin (Fn). However, integrin switches remain largely hypothetical due to a dearth of evidence for their existence, and relevance, in vivo. By using phage display in combination with the controlled deposition and extension of Fn fibers, we report the discovery of peptide-based molecular probes capable of selectively discriminating Fn fibers under different strain states. Importantly, we show that the probes are functional in both in vitro and ex vivo tissue contexts. The development of such tools represents a critical step in establishing the relevance of theoretical mechanotransduction events within the cellular microenvironment.