In both one- and two-photon bioimaging, fluorescent proteins are now commonly used. While many have been designed/engineered to have different photophysical properties, such as absorption/emission wavelength, brightness, or decreased phototoxicity/photobleaching, research continues on new variants. One promising track of research is to investigate beyond the 20 canonical amino acids to incorporate non-canonical amino acids into fluorescent proteins.1,2 One example is the work accomplished by Zhang and Ai3 where they demonstrate the capability of an amino-substituted tyrosine residue to perform a green-to-red conversion of the absorption spectra of various green fluorescent proteins. In the present work, strategies to red-shift superfolder Green Fluorescent Protein (sfGFP) are presented. To accomplish this task, time-dependent density functional theory is used to benchmark the photophysical properties of both the neutral and anionic forms of the canonical and non-canonical chromophores in isolation. We then apply Quantum Mechanical/Molecular Mechanical (QM/MM) approaches to determine the photophysical properties of sfGFP. Analogous QM/MM computations on the amino-tyrosine substituted chromophore in sfGFP are performed to examine the differences between the neutral and anionic chromophore, Z and E isomers, the barrier to isomerization, as well as the role of nearby residues and waters on the photophysical properties. (1) Salem, M. A.; Twelves, I.; Brown, A. Prediction of Two-Photon Absorption Enhancement in Red Fluorescent Protein Chromophores Made from Non-Canonical Amino Acids. Phys. Chem. Chem. Phys. 2016, 18 (35), 24408–24416. https://doi.org/10.1039/c6cp03865d. (2) Rossano-Tapia, M.; Olsen, J. M. H.; Brown, A. Two-Photon Absorption Cross-Sections in Fluorescent Proteins Containing Non-Canonical Chromophores Using Polarizable QM/MM. Front. Mol. Biosci. 2020, 7 (June). https://doi.org/10.3389/fmolb.2020.00111. (3) Zhang, S.; Ai, H. Wang. A General Strategy to Red-Shift Green Fluorescent Protein-Based Biosensors. Nat. Chem. Biol. 2020, 16 (12), 1434–1439. https://doi.org/10.1038/s41589-020-0641-7.