We started our study with the functionalization of tetramer AFYA-NH2 (1a) as model substrate. AFYA-NH2 (1a) was easily obtained by solid-phase peptide synthesis (SPPS) and allowed a good detection by HPLC. Although 1a did not contain nucleophilic side chains, the presence of the free N-terminus was already expected to give an impression of the selectivity of the method towards other nucleophiles. We chose the azidefunctionalized EBX JW-RF-010 (2a) as labeling reagent, as the azide group can be used for further functionalization via SPAAC. By simple dissolution of the peptide in non-degassed Tris buffer (10 mM, pH 8.2), and then addition of a solution of EBX 2a in DMSO, the O-VBX bioconjugate 3a could be obtained in 76% HPLC-yield after 24 hours of reaction at 37 °C (Table , entry 1). The reaction was clean and the only side products observed were N3-VBX 4 and 2-iodobenzoic acid (5), both originating from EBX decomposition. No reaction on the potentially nucleophilic N-terminus was observed. Decreasing the buffer molarity to 1 mM slowed down the bioconjugate formation, whereas increasing to 100 mM allowed to reach 96% HPLC-yield (entries 2-3). Under higher molarity, solubility issues of the peptide and higher EBX decomposition were observed (entry 4). The reaction displayed high tolerance towards buffer (entries 5-7), but additional side products were detected in PB, HEPES and PBS buffers. The pH was found to be an important factor for the efficiency of the labelling. At pH close to physiological pH (7.0), only 24% of O-VBX was formed (entry 8), whereas under more basic conditions (pH 9.0), quantitative peptide functionalization could be achieved (entry 9). Performing the reaction at room temperature slightly slowed down the labelling (entry 10). At higher temperature, the labelling remained efficient, but higher EBX decomposition was observed (entries 11-12). The developed methodology required 24 hours to reach full conversion of the peptide to the corresponding labelled product. However, decent conversion could be obtained in shorter time. After 1 hour, almost 60% conversion was achieved (entry 13), and up to 80% yield was obtained in 4 hours (entry 14).