Purpose To characterise skin barrier function in vivo at two distinct anatomic sites using minimally invasive bioengineering and biophysical tools. Methods In healthy human volunteers, the quantities of stratum corneum (SC) per unit area of skin on the forearm and forehead were quantified by gravimetric and imaging techniques. Organisation of the SC intercellular lipids was evaluated as a function of position using attenuated total reflectance infrared spectroscopy (ATR-IR). The constituents of natural moisturising factor (NMF) were extracted from tape-stripped samples of the SC and by reverse iontophoresis; 21 components were identified and quantified by liquid chromatography with mass spectrometric detection. Results SC was quantified more accurately by imaging and was significantly thinner on the forehead than on the forearm. Intercellular lipids were more disordered near the skin surface at both sites; however, throughout forearm SC, the lipids were substantially better organised than those in the forehead. Compositionally, the NMF from forearm and forehead SC was similar, but the total amount extractable from the forehead was smaller. Conclusion Taken together, the bioengineering and biophysical techniques employed demonstrate, in a complementary, objective and quantitative fashion, that SC barrier function on the forehead is less competent than that on the forearm.