The hoof plays an important role in the health and well-being of dairy animals. Consequently, mechanical properties of the hoof horn and changes in this tissue with nutrition or environmental factors are important concerns. A novel experimental approach for evaluating the mechanical behaviour of hoof horn has been developed. The process is comprised of obtaining incremental slices of hoof horn, stamping samples from selected zones of the sectioned tissue, performing uniaxial tensile tests and evaluating the mechanical response using digital image correlation (DIC). From a combination of unique methods of extraction and evaluation, the process enables hoof horn tissue to be characterized as a function of distance from the dermal-epidermal junction, within specific regions of the claw and as a function of hydration. In addition, the methods enable both the elastic and inelastic response of the tissue to be quantified. A preliminary study was performed to validate the new approach. In this manuscript the methods of evaluation are described and demonstrated through an examination of the mechanical behaviour of bovine hoof horn at two different levels of hydration.