Abstract <p>The change in the structure of Nile red after treatment with acids of different strengths (acetic, formic, and hydrochloric) was studied by Fourier-transform IR spectroscopy. The IR spectra of the dye dried after treatment with formic and hydrochloric acids showed absorption bands corresponding to the stretching vibrations of the N−H⁺ bonds of tertiary amines and O−H bonds of hydroxyl groups. The IR spectra of the dye dissolved in acetic and formic acids showed predominantly the absorption band of hydroxyl O−H bonds. Thus, the amino and carbonyl groups were found to be the most probable protonation sites in the Nile red molecule. An analysis of the UV-visible spectra of the dye in acetic and formic acids and in DMSO showed that in acetic acid, Nile red probably exists mainly in the unprotonated form, while in the stronger formic acid, a significant portion of its molecules are protonated, which is accompanied by a more complex spectrum shape.</p>