This paper describes a scatterometry approach designed by simulations for the in-line characterization of sub-wavelength sinusoidal gratings, which are formed on a transparent foil in a roll-to-roll procedure. Currently used methods are based on series of in situ measurements of the specular optical response at different incident angles or wavelengths for acquiring dimensional information on the gratings. The capability of single measurements of the first diffraction maxima at a fixed incident angle and wavelength to accurately measure the height of the sub-wavelength sinusoidal gratings is investigated in this work. The relation between the scattered powers of the diffraction maxima and the grating height is extracted from light scattering simulations, i.e., the inverse problem is solved. Optimal setup parameters for the measurement of grating heights ranging from 100 nm to 300 nm are derived from simulations. Limits of measurability and the measurement uncertainty are evaluated for different instrumentation and simulation parameters. When using laser light in the visible wavelength range, the measurement uncertainty is physically limited by the photon shot noise to the picometer range, but the systematic contributions dominate the uncertainty. As a result, the measurement uncertainty for the grating height is estimated to ≤12 nm, with a potential for <4 nm. Large-area scanning measurements performed offline and reference atomic force microscopy measurements verify the sensitivity of the presented measurement approach for identifying local variations of the spatial surface properties. Depending on the chosen detection system, sampling rates up to the MHz range are feasible, meeting the requirements of in-line process control of the roll-to-roll production process.