Noncanonical amino acids (ncAAs) have become essential tools in biocatalysis and enzyme engineering, enabling the introduction of new functional elements into proteins. This review discusses the use of ncAAs to expand the range of amino acid building blocks available for protein design, allowing for the development of enzymes with improved activity, selectivity, and stability. Genetic code reprogramming methods, such as selective pressure incorporation (SPI) and genetic code expansion (GCE), have enabled the incorporation of ncAAs into proteins, providing new insights into enzyme mechanisms and catalytic processes. These methods allow for the introduction of spectroscopic probes, covalent trapping of reactive intermediates, and modulation of noncovalent interactions, all of which enhance our understanding of enzyme function. The integration of ncAAs with laboratory evolution has led to the development of novel enzymes that utilize ncAAs as key catalytic elements. The review highlights various applications of ncAAs in enzyme design, including the study of protein structure and dynamics, the characterization of enzyme mechanisms, and the engineering of biocatalysts with enhanced properties. The use of ncAAs has also facilitated the development of enzymes with artificial regulatory elements that respond to external stimuli. Overall, the availability of ncAAs has opened up new opportunities in enzymology and biocatalysis, offering a powerful tool for the design and engineering of enzymes.Noncanonical amino acids (ncAAs) have become essential tools in biocatalysis and enzyme engineering, enabling the introduction of new functional elements into proteins. This review discusses the use of ncAAs to expand the range of amino acid building blocks available for protein design, allowing for the development of enzymes with improved activity, selectivity, and stability. Genetic code reprogramming methods, such as selective pressure incorporation (SPI) and genetic code expansion (GCE), have enabled the incorporation of ncAAs into proteins, providing new insights into enzyme mechanisms and catalytic processes. These methods allow for the introduction of spectroscopic probes, covalent trapping of reactive intermediates, and modulation of noncovalent interactions, all of which enhance our understanding of enzyme function. The integration of ncAAs with laboratory evolution has led to the development of novel enzymes that utilize ncAAs as key catalytic elements. The review highlights various applications of ncAAs in enzyme design, including the study of protein structure and dynamics, the characterization of enzyme mechanisms, and the engineering of biocatalysts with enhanced properties. The use of ncAAs has also facilitated the development of enzymes with artificial regulatory elements that respond to external stimuli. Overall, the availability of ncAAs has opened up new opportunities in enzymology and biocatalysis, offering a powerful tool for the design and engineering of enzymes.