Although tryptophan is a natural probe of protein structure, interpretation of its fluorescence emission spectrum is complicated by the presence of two electronic transitions, 1La and 1Lb. Theoretical calculations show that a point charge adjacent to either ring of the indole can shift the emission maximum. This study explores the effect of pyrrole and benzyl ring substitutions on the transitions' energy via absorption and fluorescence spectroscopy, and anisotropy and lifetime measurements. The survey of indole derivatives shows that methyl substitutions on the pyrrole ring effect 1La and 1Lb energies in tandem, whereas benzyl ring substitutions with electrophilic groups lift the 1La/1Lb degeneracy. For 5- and 6-hydroxyindole in cyclohexane, 1La and 1Lb transitions are resolved. This finding provides for 1La origin assignment in the absorption and excitation spectra for indole vapor. The 5- and 6-hydroxyindole excitation spectra show that despite a blue-shifted emission spectrum, both the 1La and 1Lb transitions contribute to emission. Fluorescence lifetimes of 10 ns for 5-hydroxyindole are consistent with a charge acceptor-induced increase in the nonradiative rate (1).