Commercial reverse osmosis (RO) membranes typically consist of an active aromatic polyamide layer (APA, thickness ∼200 nm), polymerized on a porous polysulfone support, itself attached to a polyester backing fabric. RO membranes are widely used in industrial waste-water treatments and their performances are essentially dominated by the active APA layer. However, neutral organic molecules rejections and the corresponding transfer behaviors are difficult to predict. In this work, we combined experimental structural characterizations and molecular modeling investigations on APA films, in order to improve transfer understanding at molecular level.APA layer of commercial CPA2 RO membrane and a set of free-standing APA films synthesized at four organic solvent temperatures (-20°C, 0°C, 15°C and 29°C) were investigated. Their structural properties including the morphology, topology, thickness, roughness, void fraction, effective density, dense layer density and chemical structure were characterized via various techniques such as Field Emission Scanning Electron Microscopy (FE-SEM), Atomic Force Microscopy (AFM), Profilometry, Spectroscopic Ellipsometry, Dynamic water sorption (DVS), X-ray photoelectron spectra (XPS) and Atomic force microscopy-based infrared spectroscopy (AFM-IR). Both film types had a multi-level topological structure with a dense base upon which generates: a valley-ridge structure for CPA2 or a chimney-like structure for synthesized samples, for which thickness and chimneys size increased with temperature. Based on the obtained average void fraction of 35% and the apparent volumetric density of 0.81 g·cm-3, the density of the dense regions of CPA2 APA layer was calculated at 1.25 g·cm-3 when dry and 1.48 g·cm-3 when hydrated. This completes the very few experimental density values found for the dense part of commercial APA films. With the help of a new set of chemical structure descriptors, the chemical depth-heterogeneity of CPA2 APA layer was investigated. The film synthesized at -20°C performed a remarkable water uptake of 65% at 91%RH, which might be attributed to its orderly distributed and small-size chimney morphology on top-surface.All-atoms molecular models of APA polymer were constructed by forming amide bonds between trimesoyl chloride (TMC) and m-phenylenediamine (MPD) with a set of initial MPD:TMC monomers ratios, varying from 0.25 to 5. The purposes were, on one hand, to mimic APA’s possible depth-dependent heterogeneous structures with boxes of different cross-linking degrees; on the other hand, to understand the effect of different initial monomer proportions during the IP on APA film’s structure. Several steps were necessary in order to obtain equilibrated samples, including a very long (several microseconds) constant stress and temperature molecular simulation. Final MPD:TMC (connected) ratios ranging from 0.68 to 2.61 were observed, corresponding to systems with acyl/amine group connectivity degrees at 45%/100% or 99%/57%, respectively. These results were consistent with chemical structure evaluation of real APA films. A similar density around 1.26 g·cm-3 were observed for all systems, consistent with the experimental density of CPA2 APA.Water sorption isotherms were computed using Monte Carlo method in osmotic ensemble for several simulated polymer matrixes and were obtained via DVS for experimental layers. At low water activity, water sorption behaviors of simulated polymers were in agreement with experimental data, validating the overall simulation methodology. At high water activity, water absorption was underestimated by molecular simulations. This could be attributed to the existence of void space in real APA films. No significant swelling was observed in simulations, which was in agreement with our experimental results for CPA2.