Advances in fabrication have allowed tissue engineers to better mimic complex structures and tissue interfaces by designing nanofibrous scaffolds with spatially-graded material properties. However, the nonuniform properties that grant the desired biomechanical function also make these constructs difficult to characterize. In light of this, we developed a novel procedure to create graded nanofibrous scaffolds and determine the spatial distribution of their material properties. Multi-layered nanofiber constructs were synthesized, controlling spatial gradation of the stiffness to mimic the soft tissue gradients found in tendon or ligament tissue. Constructs were characterized using uniaxial tension testing with Digital Image Correlation (DIC) to measure the displacements throughout the sample, in a non-contacting fashion, as it deformed. Noise was removed from the displacement data using Principal Component Analysis (PCA), and the final de-noised field served as the input to an inverse elasticity problem whose solution determines the spatial distribution of the Young's modulus throughout the material, up to a multiplicative factor. Our approach was able to construct, characterize, and determine the spatially-varying moduli, in four electrospun scaffolds, highlighting its great promise for analyzing tissues and engineered constructs with spatial gradations in modulus, such as those at the interfaces between two disparate tissues (e.g., myotendinous junction, tendon- and ligament-to-bone entheses).