Abstract: A method for site-specific modification of protein molecules includes providing a protein molecule having at least first and second non-terminal Cys residues at respective first and second sites within the protein molecule, providing a blocking molecule that is suitable to interact with the protein molecule to selectively block the second non-terminal Cys residue to prevent modifications of the second non-terminal Cys residue while leaving the first non-terminal Cys residue unblocked, and providing a Cys-modifying molecule that is suitable to modify the first non-terminal Cys residue. The blocking molecule according to an embodiment of the invention may be a protein molecule, DNA, an aptamer or synthetic organic molecules. The Cys-modifying molecule according to an embodiment of the invention may be a fluorophore, biotin, spin label, a sugar or a non-natural amino acid analogue.