One of the most promising methods for labeling biomolecules in living systems is the use of click chemistry, a set of highly efficient and selective reactions applicable in bioorthogonal processes.1 This study focuses on the application of the nitrile-aminothiol click reaction (NAT) for peptide and protein tagging, achieved by incorporating unnatural amino acids with reactive nitrile functionalities, such as cyanopyridylalanine and dicyanovinyl-tryptophan derivatives, into target proteins to enable precise conjugation with aminothiol probes.2 Cyanopyridylalanines can be genetically encoded into peptides and proteins and they react with aminothiol compounds under physiological conditions without requiring added catalyst.3 We have developed novel fluorogenic 1,2-aminothiol probes based on naphthalimide and BODIPY fluorophores for efficient protein labeling through the nitrile-aminothiol click reaction. Additionally, we synthesized dicyanovinyl-indole derivatives featuring reactive nitrile functionalities, specifically designed to react with aminothiol moieties in click chemistry to evaluate the changes in fluorescent properties. Both the 1,2- aminothiol and nitrile fluorophores exhibit green fluorescence upon the click reaction, with the resulting click products showing a "turn-on" fluorescence response. This demonstrates that these tools can facilitate bioimaging applications in live cells and offer a sensitive and selective approach for peptide and protein labeling.