The work of adhesion and work of separation are characteristic properties of a contact interface that describe the amount of energy per unit area required to adhere or separate two contacting substrates, respectively. In this work, the authors present experimental and data analysis procedures that allow the contact interface between a soft synthetic tissue and a smooth or micropatterned poly(dimethylsiloxane) (PDMS) substrate to be characterized in terms of these characteristic parameters. Because of physical geometry limitations, the experimental contact geometry chosen for this study differs from conventional test geometries. Therefore, the authors used finite element modeling to develop correction factors specific to the experimental contact geometry used in this work. A work of adhesion was directly extracted from experimental data while the work of separation was estimated on the basis of experimental results. These values are compared to other theoretical calculations for validation. The results of this work indicate that the micropatterned PDMS substrate significantly decreases both the work of adhesion and work of separation as compared to a smooth PDMS substrate when in contact with a soft synthetic tissue substrate.