For the first time, the changes in autofluorescence spectra of ex vivo rat skin have been experimentally investigated using the combination of fluorescence spectroscopy and optical immersion clearing. The glucose, glycerol and propylene glycol solutions were used as clearing agents. The optical clearing was performed from the dermal side of skin imitating the in vivo injection of clearing agent under the dermal layers. In this contribution, the common properties of autofluorescence variation during optical immersion clearing were determined. The tendency of autofluorescence signal to decrease with reduction of scattering in tissue was noticed and discussed in detail. However, the differences in the shape of spectral curves under application of different clearing agents showed that optical clearing affects the autofluorescence properties of tissue differently depending on the type of clearing liquid. The results obtained are useful for the understanding of tissue optical clearing mechanisms and for improving techniques such as fluorescence spectroscopy.