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We have developed pLink, software for data analysis of cross-linked proteins coupled with mass-spectrometry analysis. pLink reliably estimates false discovery rate in cross-link identification and is compatible with multiple homo-or hetero-bifunctional cross-linkers. We validated the program with proteins of known structures, and we further tested it on protein complexes, crude immunoprecipitates and whole-cell lysates. We show that it is a robust tool for protein-structure and protein-protein–interaction studies.