Chemical cross-linking, followed by identification of the cross-linked residues, is a powerful approach to probe the topologies and interacting surfaces of protein assemblies. In this work, we demonstrate a new bioinformatic approach using multiple program modules within the software package ‘Protein Prospector’ that greatly facilitates the discovery of cross-linked peptides in chemical cross-linking studies. Examples are given for how this approach has been used for defining interfaces in heterodimeric and homodimeric protein complexes, both of which provide results in close agreement with crystal structures, verifying the reliability of the approach.