The Electrospinning and Stamp-thru-mold (ESTM) technique, an integrated fabrication process which incorporates the versatility of the electrospinning process for nanofiber fabrication with the non-lithographic patterning ability of the stamp-thru-mold process is introduced. In-situ multilayer stacking of orthogonally aligned nanofibers, ultimately resulting in a nanoporous membrane, has been demonstrated using orthogonally placed collector electrode pairs and an alternating bias scheme. The pore size of the nanoporous membrane can be controlled by the number of layers and the deposition time of each layer. Non-lithographic patterning of the fabricated nanoporous membrane is then performed by mechanical shearing using a pair of pre-fabricated micromolds. This patterning process is contamination free compared to other photo lithographical patterning approaches. The ability to pattern on different substrates has been tested with and without oxygen plasma surface treatment. In vitro tests of ESTM poly-lactic-co-glycolic acid (PLGA) nanofibers verify the biocompatibility of this process. Simulation by the COMSOL Multiphysics tool has been conducted for the analysis of electrospun nanofiber alignment.