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The growing interest in the production of biofuels has motivated numerous studies on separation techniques that allow the separation/concentration of organics produced by fermentation, improving productivity and performance. In this work, the preparation and characterization of new butanol-selective membranes was reported. The prepared membranes had a hollow fiber configuration and consisted of two dense selective layers: a first layer of PEBA and a second (outer) layer of PDMS. The membranes were tested to evaluate their separation performance in the selective removal of organics from a synthetic ABE solution. Membranes with various thicknesses were prepared in order to evaluate the effect of the PDMS protective layer on permeant fluxes and membrane selectivity. The mass transport phenomena in the pervaporation process were characterized using a resistances-in-series model. The experimental results showed that PEBA as the material of the dense separating layer is the most favorable in terms of selectivity towards butanol with respect to the other components of the feed stream. The addition of a protective layer of PDMS allows the sealing of possible pinholes; however, its thickness should be kept as small as possible since permeation fluxes decrease with increasing thickness of PDMS and this material also has greater selectivity towards acetone compared to other feed components.