Voltage-clamp fluorometry is a powerful tool to examine conformational changes in ligand- and voltage-gated ion channels. In particular, the technique has provided a wealth of information on electrophysiologically silent conformational changes. However, the many studies in the field using this technique were essentially limited to solvent-accessible domains of the ion channels, as it uses the covalent modification of an introduced cysteine side chain with an environmentally sensitive fluorophore. This approach has left large regions of membrane proteins off limits. We therefore aim to synthesze novel fluorescent amino acids that are exquisitely sensitive to their local dielectric environment and can be incorporated at virtually any position within the ion channel. This would be a tremendous step forward in the field, as it would enable us not only to address the question of conformational changes within the protein but also would open up new avenues to study ion channel physiology and trafficking. Unnatural amino acid side chains with fluorescent properties have been introduced into ion channels before, but progress was hampered by significant shortcomings: the amino acid was either not sensitive to its environment or expression levels were at least an order of magnitude too low to observe macroscopic fluorescent changes in vivo. Here we use our optimized in vivo nonsense suppression method to generate ion channels carrying (nonfluorescent) amino acid side chains at high enough expression levels to observe macroscopic fluorescence changes when using conventional cysteine-linked dyes. This is an important milestone as it confirms that the technique is capable of generating high enough surface expression to observe macroscopic fluorescent changes that report on conformational changes in ion channels. Furthermore, we provide experimental evidence for the successful incorporation of fluorescent amino acids such as the nitroaromatic amino acid N-methyl-amino-7-nitroben-2-oxa-1,3-diazole (NBD), which is sensitive to the local dielectric environment and can be expressed at high levels using our optimized in vivo nonsense suppression method. These results provide a significant forward towards observing macroscopic fluorescence changes from an unnatural fluorescent amino acid incorporated in an ion channel expressed in vivo.