Subtiligase-catalyzed peptide ligation is a powerful approach for site-specific protein bioconjugation, synthesis and semisynthesis of proteins and peptides, and chemoproteomic analysis of cellular N termini. Here, we provide a comprehensive review of the subtiligase technology, including its development, applications, and impacts on protein science. We highlight key advantages and limitations of the tool and compare it to other peptide ligase enzymes. Finally, we provide a perspective on future applications and challenges and how they may be addressed.