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Diazo peptides have been described earlier, however, due to their high reactivity have not been broadly used until today. Here, we report the preparation, properties, and applications of chemically stable internal diazo peptides. Peptidyl phosphoranylidene‐esters and amides were found to react with triflyl azide primarily to novel 3,4‐disubstituted triazolyl‐peptides. Nonaflyl azide instead furnished diazo peptides, which are chemically stable from pH 1–14 as amides and from pH 1–8 as esters. Thus, diazo peptides prepared by solid phase peptide synthesis were stable to final deprotection with 95 % trifluoroacetic acid. Diazo peptides with the recognition sequence of caspase‐3 were identified as specific, covalent, and irreversible inhibitors of this enzyme at low nanomolar concentrations. A fluorescent diazo peptide entered living cells enabling microscopic imaging and quantification of apoptotic cells via flow cytometry. Thus, internal diazo peptides constitute a novel class of activity‐based probes and enzyme inhibitors useful in chemical biology and medicinal chemistry. [ABSTRACT FROM AUTHOR]