A method for visual and quantitative assessment of underpacking in the protein core. RosettaHoles generates a set of spherical cavity balls that fill the empty volume between atoms in the protein interior. RosettaHoles2 features a packing measure more rapidly computable, accurate and physically transparent, as well as a new validation score intended for structures submitted to the Protein Data Bank. The differential packing measure is parameterized to maximize the gap between computationally generated and experimentally determined X-ray structures, and can be used in refinement of protein structure models.