Circular peptides have long been sought after as scaffolds for drug design as they demonstrate protein-like properties in the context of small, constrained peptides. Traditional routes toward the production of cyclic peptides rely on synthesis or semisynthetic methods, which restrict their use as platforms for the production of large, structurally diverse chemical libraries. Here, we discuss the biosynthetic routes toward the N-C macrocyclization of linear peptide precursors, specifically, those transformations that are catalyzed by peptidases. While canonical peptidases catalyze the proteolysis of linear peptides, the biosynthetic macrocyclases couple proteolytic cleavage with cyclization to produce macrocyclic compounds. In this Perspective, we explore the different structural features that impart on each of these biosynthetic proteases the distinct ability to perform macrocyclization and focus on their potential use in biotechnology.