We have demonstrated that both multiphoton polarization and generalized polarization (GP) microscopy may be combined to characterize the structural changes of intercellular lipids in skin. Both polarization and GP (at 440- and 490-nm emission) images obtained by analysis of Laurdan fluorescence suggest that the treatment of oleic acid results in a skin surface with a more random packing of lipid molecules, which allows easier water penetration. Our results show that combined polarization and GP microscopy can be used to characterize the physical and chemical changes in biological structures.