This study presents the design and implementation of a prototype device that interfaces with commercial atomic force microscopy (AFM) instruments to accurately measure ex vivo, regional, passive elasticity measurements of the murine myocardium in three-dimensions (3D). The constructed prototype has a translation stage base allows fine adjustment of planar location at the micrometer level of accuracy. Four degrees of motional freedom are allowed in total, permitting fine, independent rotations of the murine heart (oriented at right-angles with respect to its normal mode of operation) at 2–5° increments, along its two axes of symmetry. Successful mounting of the constructed prototype was realized on the AFM's base platform, with Z-positioning maintained through the instrument's electronics. Measurements of normal epicardial tissue elasticity in one excised male C57BL/6 heart led to a mean value (over 15 epicardial locations) of 51.8 ± 5.4 kPa. A flexible, versatile, and low-cost prototype assembly was designed, implemented, and tested to conduct epicardial elasticity measurements in a semi-automated manner, using AFM. The epicardial elasticity values are within expected, published ranges for the normal myocardium of 10–55 kPa. Efforts are ongoing to allow 3D regional elasticity measurements at finer resolution with potential usefulness to studies of genetically altered mice.