Various micro-devices have been used to assess single cell mechanical properties. Here, we designed and implemented a novel, mechanically actuated, two dimensional cell culture system that enables a measure of cell stiffness based on quantitative functional imaging of cell-substrate interaction. Based on parametric finite element design analysis, we fabricated a soft (5 kPa) polydimethylsiloxane (PDMS) cell substrate coated with collagen-I and fluorescent micro-beads, thus providing a favorable terrain for cell adhesion and for substrate deformation quantification, respectively. We employed a real-time tracking system that analyzes high magnification images of living cells under stretch, and compensates for gross substrate motions by dynamic adjustment of the microscope stage. Digital image correlation (DIC) was used to quantify substrate deformation beneath and surrounding the cell, leading to an estimate of cell stiffness based upon the ability of the cell to resist the applied substrate deformation. Sensitivity of the system was tested using chemical treatments to both "soften" and "stiffen" the cell cytoskeleton with either 0.5 μg/ml Cytochalasin-D or 3% Glutaraldehyde, respectively. Results indicate that untreated osteosarcoma cells (SAOS-2) exhibit a 1.5 ± 0.7% difference in strain from an applied target substrate strain of 8%. Compared to untreated cells, those treated with Cyochalasin-D passively followed the substrate (0.5 ± 0.5%, p < 0.001), whereas Glutaraldehyde enhanced cellular stiffness and the ability to resist the substrate deformation (2.9 ± 1.6%, p < 0.001). Nano-indentation testing showed differences in cell stiffness based on culture treatment, consistent with DIC findings. Our results indicate that mechanics and image analysis approaches do hold promise as a method to quantitatively assess tensile cell constitutive properties.