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Among all methods available for the preparation of multifunctional nanostructured composite materials with remarkable functional properties, Layer-by-Layer (LbL) assembly is currently one of the most widely used techniques due to its environmental friendliness, its ease of use and its versatility in combining a plethora of available colloids and macromolecules into finely tuned multicomponent architectures with nanometer scale control. Despite the importance of these systems in emerging technologies, their nanoscopic 3D structure, and thus the ability to predict and understand the device performance, is still largely unknown. In this article, we use neutron scattering to determine the average conformation of individual deuterated polyelectrolyte chains inside LbL assembled films. In particular, we determine that in LbL-films composed of poly(sodium 4-styrenesulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) multilayers prepared from 2 M sodium chloride solutions the PSS chains exhibit a flattened coil conformation with an asymmetry factor of around seven. Albeit this highly non-equilibrium state of the polymer chain, its density profiles follow Gaussian distributions occupying roughly the same volume as in the bulk complex., (© 2023. The Author(s).)