<p>Cross-linking mass spectrometry has become a powerful technique for identifying protein interactomes and studying protein structures. We report the development of homo-bi-functional photo-activatable (BFPA) cross-linkers for cross-linking mass spectrometry. These cross-linkers theoretically react with any-to-any amino acid, overcoming limitations in amino acid reactivity. Different fragmentation energies and cross-linking conditions were tested, and the false discovery rate was benchmarked against a non-cross-linked sample and a false protein sequence search across different search engines. BFPA cross-linkers identified cross-links with better overall agreement with high resolution protein structures compared to randomized cross-links and compared to other commonly used cross-linkers. Different cross-link sites were identified by BFPA cross-linkers across bovine serum albumin, human importin complex and dengue protein complex, demonstrating its wide applicability to different protein complexes. BFPA have the potential to serve as complementary tools to current cross-linkers to expand the coverage of cross-linking mass spectrometry experiments.</p>