Heteroaromatic stacking interactions help stabilize protein tertiary structure and are important in drug binding, supramolecu-lar chemistry, and materials science. Although diverse computational and experimental approaches have been utilized to study these interactions, a broadly useful protein–ligand model system has yet to emerge, despite laudable efforts by Diederich and co-workers in this vein. Here we studied thirty synthetic ligands that present diverse heteroarene probes for stacking between symmetry-related tyrosine residues at the dimer interface of procaspase-6. We demonstrate crystallograph-ically that stacking geometries are highly conserved across the ligand test set and show with high-accuracy computations that differences in ligand binding free energies are primarily attributable to the relative strength of the stacking interactions. The empirical results are discussed in light of recent computational studies of these interactions, including the effects of torsional strain, heteroarene tautomeric state, and co-axial orientation of stacking groups. Overall, this study provides an extensive dataset of empirical and high-level computed binding energies in a versatile new protein–ligand system highly amenable to studies of other intermolecular interactions.