Purpose <p>Enzymatic reactions offer many advantages for hydrogel synthesis and modification, due to their gentle reaction conditions, biocompatibility, and diversity of substrates.</p> Methods <p>In this review, we examine the current body of literature through databases such as Google Scholar, PubMed, and Web of Science.</p> Results <p>Various enzyme classes have been utilized for hydrogel assembly and disassembly, including transglutaminases, oxidoreductases, transpeptidases, and proteinases. The enzymatic substrates can be readily included in peptide precursors and/or appended onto synthetic polymers. We discuss the benefits and limitations of each system, with a focus on ease of use/synthesis, accessibility, and financial considerations.</p> Conclusion <p>Enzymes are frequently utilized to modify both natural and synthetic biomaterials. For developing more advanced, stimuli-responsive platforms, “biologically invisible” enzymes such as sortases should be leveraged to not interfere with native processes and/or the mammalian proteome.</p> Lay Summary <p>Enzymes, proteins that act as biological catalysts, are an important tool for making and breaking down hydrogels, or water-swollen polymeric networks, for various biomedical applications. In particular, these techniques have seen great usage for modeling the tissue environment for lab-based assays.</p> Graphical Abstract <p></p>