The site-specific introduction of unnatural amino acids (UAAs) has been demonstrated to be a useful tool in protein engineering. Moreover, the incorporation of a UAA into a protein has become feasible with the increased commercial availability of UAAs and robust expression plasmids. In addition to the ease of incorporation, the concepts utilized in the incorporation of a UAA are ideal to illustrate key concepts involved in protein translation and structure-function relationships. Consequently, a novel experiment has been developed for the incorporation of various UAAs into the GFP fluorophore to alter spectral properties. This experiment provides students with a hands-on approach for transformation, protein expression and purification, and structure-function analysis.