5-Hydroxytryptophan is a non-natural amino acid that has attracted a lot of recent interest as a fluorescent probe of protein structure, dynamics, and function. We have investigated its fluorescence in various protein environments, using a decoupled quantum mechanics/molecular mechanics approach. Classical, all-atom molecular dynamics simulations of several proteins containing single tryptophans were performed for both the wild-type and the 5-hydroxy derivatives. The excited state of the fluorophore was described using parameters from complete active space self-consistent field calculations. Time-dependent density functional theory calculations on 5-hydroxytryptophan and a significant portion of its explicit immediate surrounding environment, sampled by the simulations, show that the emission energies of 5-hydroxytryptophan shift, depending on the strength of hydrogen bonding and pi-pi stacking interactions. This quantitative description of how the fluorescence responds to different protein environments should enhance the insight that fluorescence studies using 5-hydroxytryptophan can provide at a molecular level.