The effect of bicelles formed by dipalmitoylphosphatidylcholine (DPPC)/dihexanoylphosphatidylcholine (DHPC) on stratum corneum (SC) lipids was studied by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy at different temperatures. Analysis of the lipid organization in terms of chain conformational order and lateral packing shows that the use of bicelles hampers the fluidification of SC lipids with temperature and leads to a lateral packing corresponding to a stable hexagonal phase. Grazing incidence small- and wide-angle X-ray scattering (GISAXS and GIWAXS) techniques confirm these results and give evidence of higher lamellar order after treatment with these bicelles. Additionally, the effects of DPPC/DHPC and dimyristoylphosphatidylcholine (DMPC)/DHPC bicelles at different SC depths were compared. The combination of ATR-FTIR spectroscopy and the tape-stripping method was very useful for this purpose.