Modulating PPIs has been regarded as a promising strategy for the next-generation therapeutics. Medium-size peptide drugs are highly promising for targeting PPIs due to their potential target specificity as big biologics but with smaller sizes. To enhance the properties of bioactive peptide sequences, chemical modifications of specific residues and macrocyclizations on the peptides are of great importance. In this context, hypervalent iodine reagents (HIRs), especially EthynylBenziodoXolone (EBX) derivatives have emerged as useful tools for selective residue modifications. Intrigued by the excellent reactivity of EBXs, we first focused on the introduction of highly electrophilic EBX reagent onto multifunctional peptides. This was achieved through the selective amidation of lysine or N-termini with a bifunctional EBX reagent. The resulting peptide-EBX conjugates served as effective intermediates for subsequent peptide cross-coupling through S-alkynylation, S-alkenylation, and decarboxylative alkynylation. Moreover, we developed a novel peptide macrocyclization via decarboxylative alkynylation enabled by photoredox catalysis. The rigidity of those alkyne-embedded cyclic peptides exhibited promising binding affinity to the Keap1-Nrf2 binding site. Despite the simplicity and efficiency of introducing EBXs, the stability of peptide-EBXs has hindered their broader application. The stability of peptide-EBXs was enhanced by modifying the EBX backbone to bis-CF3 alcohol (EBx). With the second generation of peptide-EBxs, we developed another peptide macrocyclization targeting tryptophan enabled by gold(I) catalysis. The intramolecular Trp C2 C-H alkynylation extended the aromatic system of Trp, yielding a cyclic peptide with a fluorescent cyclic linker. We underscored the utility of these cyclic peptides by applying them as fluorescent probes for live-cell imaging. To further explore the potential of EBx handle for bioorthogonal peptide macrocyclization. We introduced the EBx core onto various amine-based amino acid building blocks. Those EBx amino acid building blocks displayed excellent compatibility with both solid phase peptide synthesis (SPPS) and solution-phase peptide synthesis (SPS), facilitating the incorporation of EBxs at specific sites within peptides. The formed peptide-EBxs can further undergo various macrocyclizations with different native amino acid residues, resulting in structurally and topologically diverse cyclic peptides. Finally, we explored the incorporation of functional peptides onto carbazolyl cyanobenzene-based (CzPN) dyes, following a serendipitous discovery during the development of peptide macrocyclization via decarboxylation. The C-terminal radical generated via peptide C-terminal decarboxylation can be trapped by CzPN. Building upon this initial finding, we developed two efficient bioconjugation methods for introducing the CzPN dye. One through C-terminal decarboxylative arylation, enabling the introduction of functional peptides onto a structurally diverse range of CzPN derivatives. The other involves the SnAr reaction of the 3CzFPN precursor, offering a more efficient dye introduction onto cysteines. We highlighted the utility of these peptide-CzPN conjugates by applying them to decarboxylative alkynylation and thiol-ene reactions. Most importantly, these peptide-CzPN conjugates facilitate the efficient excitation of diazirine and phenyl azide for photo proximity labeling.