In summary, we have reported a chemo-and site-selective tyrosine bioconjugation methodology using hypervalent iodine reagents. Under mild and biocompatible conditions, tyrosine residues reacted with EBX reagents to generate stable O-VBX bioconjugates. The high functional group tolerance of the conjugation method allowed to selectively label a broad range of bioactive peptides, including the natural product jagaricin (3w). Preliminary results were obtained with selected proteins and an antibody. The generated O-VBX conjugates could be further functionalized orthogonally by palladium-catalyzed Suzuki-Miyaura cross-coupling and azide-alkyne cycloaddition, providing a wide range of labelled biomolecules. The potential of the doubly-orthogonal functionalization was demonstrated in a cellular uptake experiment, which further establishes that the functionalized streptavidin retains its structural integrity and activity to bind biotin, a crucial feature for applications in chemical biology. This work further highlights the potential of hypervalent iodine reagents in biomolecule labeling. Moreover, the high tyrosine selectivity described compares well with existing methods for O-functionalization of tyrosine residues, in which labelling of other nucleophilic amino acids such as lysine and histidine has often been reported.