This paper reports on the development of polymer electrolyte membranes (PEMs) based on sulfonated polysulfone for application in a DMFC mini-stack operating at room temperature in passive mode. The sulfonated polysulfone (SPSf) with two degrees of sulfonation (57 and 66%) was synthesized by a well-known sulfonation process. SPSf membranes with different thicknesses were prepared and investigated. These membranes were characterized in terms of methanol/water uptake, proton conductivity, and fuel cell performance in a DMFC single cell and mini-stack operating at room temperature. The study addressed (a) control of the synthesis of sulfonated polysulfone, (b) optimization of the assembling procedure, (c) a short lifetime investigation and (d) a comparison of DMFC performance in active-mode operation vs. passive-mode operation. The best passive DMFC performance was 220mW (average cell power density of about 19mWcm−2), obtained with a thin SPSf membrane (70μm) at room temperature, whereas the performance of the same membrane-based DMFC in active mode was 38mWcm−2. The conductivity of this membrane, SPSf (IEC=1.34mequiv.g−1) was 2.8×10−2 Scm−1. A preliminary short-term test (200min) showed good stability during chrono-amperometry measurements.