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Structured metal thin-film electrodes are heavily used in electrochemical assays to detect a range of analytes including toxins, biomarkers, biological contaminants and cell cultures using amperometric, voltammetric and impedance-based (bio)sensing strategies as well as separation techniques such as dielectrophoresis. Over the last decade, thin-film electrodes have been fabricated onto various durable and flexible substrates including glass, silicon and polymers. However, the combination of thin-film technology with porous polymeric substrates frequently used for biochips often results in limited resolution and poor adhesion of the metal thin-film, thus severely restricting reproducible fabrication and reliable application in e.g. organ-on-a-chip systems. To overcome common problems associated with micro-structured electrode manufacturing on porous substrates, we have optimized a bi-layer lift-off method for the fabrication of thin-film electrodes on commercial porous polyester membranes using a combination of LOR3A with AZ5214E photoresists. To demonstrate practical application of our porous electrode membranes for trans-epithelial electrical resistance measurements a tetrapolar biosensing set-up was used to eliminate the artificial resistance of the porous polymer membrane from the electrochemical recordings. Furthermore, barrier resistance of Bewo trophoblast epithelial cells was compared to a standard Transwell assay readout using a EVOM2 volt-ohm meter. • Bi-layer photo resist lift-off yields resolution down to 2.5 μm. • Argon Plasma-assisted lift-off results in improved adhesion of gold thin films and eliminates the need for chromium adhesion layers. • Membrane electrodes can be used for elimination of the porous membrane resistance during tetra-polar epithelial resistance measurements. Method name: Bi-layer photoresist lift-off, Keywords: Micro fabrication, Track-etched membranes, Metal thin-film electrodes, Bi-layer lift-off