Negamycin is a hydrazide-containing antibiotic characterized by an unusual N–N bond. It exhibits broad-spectrum activity against both Gram-positive and Gram-negative bacteria by inhibiting translation. In addition, its ability to induce stop codon read-through makes it a promising therapeutic candidate for the treatment of genetic diseases caused by premature termination codons. Negamycin is produced by the actinomycete Kitasatospora purpeofusca, yet its biosynthetic gene cluster and biosynthetic pathway remain uncharacterized. To uncover the molecular basis of negamycin biosynthesis, we employ comparative proteomics by analyzing differences between the wild-type strain and a negamycin-deficient mutant. This approach aims to identify proteins potentially involved in the biosynthetic process.