In this work, the interfacial behavior of two different aqueous two-phase systems (ATPSs) was analyzed. One ATPS is formed by the dissolution of polyethylene glycol (PEG) and dextran T40 in water, and the second ATPS is formed by a hyperbranched polyesteramide (HB) and dextran T40. The interfacial behavior of both ATPSs was investigated experimentally and theoretically. As a thermodynamic model, the lattice cluster theory combined with a lattice Wertheim model was used. This thermodynamic model was combined with density gradient theory to model the interfacial properties. It was found that the interfacial tension, which was measured by the spinning drop method, of the ATPS containing the HB was lower than the interfacial tension of the ATPS containing the PEG. Moreover, the mass transfer of the phase forming components across the interface was measured and modeled. To measure the mass transfer, a Nitsch cell was used. The mass transfer was modeled by the instationary density gradient theory. It was found that the mobility coefficient of dextran could be transferred from one system to the other one.