Acridonylalanine (Acd) is a fluorescent unnatural amino acid that can site specifically label a protein of interest during protein translation. Acd’s incorporation relies on an evolved tRNA synthetase that specifically charges Acd to its cognate tRNA. As the protein mRNA sequence is being translated by the ribosome and an amber stop codon is present, the tRNA will add Acd to the growing protein chain. This labeling strategy has aided several previous biophysical studies involving fluorescence polarization (FP) and Förster resonance energy transfer (FRET) measurements, but to date was limited to solely E. coli expressed proteins. This thesis work will continue to explore Acd’s use in in vitro measurements from E. coli expressed proteins. Mainly this will focus of using Acd as a FRET and FP probe for studying protein-protein interactions in the context of the SOS antibiotic resistance pathway, and α-synuclein aggregation. In addition, I will present the successful incorporation of Acd into mammalian cells. This collaborative effort resulted in the ability to monitor protein localization using confocal microscopy and through fluorescence lifetime imaging microscopy (FLIM). Furthermore, I will demonstrate our synthetic efforts to red-shift Acd’s emission and our preliminary efforts to incorporate these red-shifted Acd derivatives using a newly evolved synthetase. While our current efforts to incorporate Acd derivatives are unsuccessful, we have a crystal structure of the synthetase and Acd that can guide our future enzyme evolution strategy.