For the first time, the thickness and refractive index of monolayers at the air/water interface have simultaneously been determined by null ellipsometry. Separation of refractive index from film thickness has been achieved by highly precise measurements of the two ellipsometric angles Psi and Delta. In the solid state, film thicknesses of arachidic acid and valine gramicidin A obtained by ellipsometry are comparable with those obtained by the X-ray techniques. For arachidic acid in the condensed state, our results suggest that only the thickness of the hydrophobic moiety is measured. When highly hydrated, the thickness of the polar headgroup is not detected. This is presumably due to its refractive index being the same as that of the bulk water; hence, the calculated film thickness corresponds to the thickness of the hydrophobic part only. As molecular area is reduced, the polar headgroup gradually loses hydration water molecules causing its refractive index profile to become different from that of the bulk water. Our results suggest that the measurable thickness of the film-forming molecules increases as the degree of dehydration of the headgroup increases.