A series of steady-state fluorescence anisotropy experiments has been performed to demonstrate the presence of a deprotonated open side chain form of warfarin in organic environments. We explain the observed emission-wavelength-dependent anisotropy of warfarin in ethanol, 2-propanol, and acetonitrile due to the coexistence of neutral isomers and deprotonated open side chain forms displaying different fluorescence decay kinetics. To investigate solvent-solute interactions in more detail, a series of molecular dynamics simulations was performed to study warfarin solvation and to predict the time scale of rotational diffusion displayed by this compound. Predictions obtained provide an explanation for the nonzero values in anisotropy observed for neutral isomers of warfarin associated with the short fluorescence lifetime (tau < 0.1 ns) and for an approximately zero anisotropy observed for the deprotonated open side chain form, which is associated with the longer fluorescence lifetime (tau = 0.5-1.6 ns). Finally, we address the potential use of fluorescence anisotropy for an increased understanding of the structural diversity of warfarin in protein binding pockets.