Fusion proteins play an essential role in the biosciences but suffer from several key limitations, including the requirement for N-to-C terminal ligation, incompatibility of constituent domains, incorrect folding, and loss of biological activity. This perspective focuses on chemical and enzymatic approaches for the post-translational generation of well-defined protein-protein conjugates, which overcome some of the limitations faced by traditional fusion techniques. Methods discussed range from chemical modification of nucleophilic canonical amino acid residues to incorporation of unnatural amino acid residues and a range of enzymatic methods, including sortase-mediated ligation. Through summarizing the progress in this rapidly growing field, the key successes and challenges associated with using chemical and enzymatic approaches are highlighted and areas requiring further development are discussed.