The site-specific incorporation of cross-linkable designer amino acids into proteins is useful for covalently bonding protein complexes upon exposure to light. This technology can be used to study networks of protein-protein interactions in living cells; however, to date it has only been applicable for use with a narrow range of cell types, due to the limited availability of plasmid-based transfection protocols. In the present study, we achieved adenovirus-based expression of a variant of an archaeal pyrrolysyl-tRNA synthetase and UAG-recognising tRNA pair, which was used to incorporate unnatural amino acids into proteins at sites defined by in-frame UAG codons within genes. As such, the site-specific photo-cross-linking method is now applicable to a wide variety of mammalian cells. In addition, we repositioned the reactive substituent of a useful photo-cross-linker, N-epsilon-(para-trifluoromethyl-diazirinyl-benzyloxycarbonyl)-l-lysine (pTmdZLys), to the meta position, which improved its availability at low concentration. Finally, we successfully applied this system to analyse the formation of a protein complex in response to a growth signal in human cancerous cells and human umbilical vein endothelial cells. This adenovirus-based system, together with the newly designed cross-linkable amino acid, will facilitate studies on molecular interactions in various cell lines of medical interest.