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The basal lamina or basement membrane (BM) is a key physiological system that participates in physicochemical signaling between tissue types. Its formation and function are essential in tissue maintenance, growth, angiogenesis, disease progression, and immunology. In vitro models of the BM (e.g., Boyden and transwell chambers) are common in cell biology and lab-on-a-chip devices where cells require apical and basolateral polarization. Extravasation, intravasation, membrane transport of chemokines, cytokines, chemotaxis of cells, and other key functions are routinely studied in these models. The goal of the present study was to integrate a semipermeable ultrathin polymer membrane with precisely positioned pores of 2 μm diameter in a microfluidic device with apical and basolateral chambers. We selected poly(l-lactic acid) (PLLA), a transparent biocompatible polymer, to prepare the semipermeable ultrathin membranes. The pores were generated by pattern transfer using a three-step method coupling femtosecond laser machining, polymer replication, and spin coating. Each step of the fabrication process was characterized by scanning electron microscopy to investigate reliability of the process and fidelity of pattern transfer. In order to evaluate the compatibility of the fabrication method with organs-on-a-chip technology, porous PLLA membranes were embedded in polydimethylsiloxane (PDMS) microfluidic devices and used to grow human umbilical vein endothelial cells (HUVECS) on top of the membrane with perfusion through the basolateral chamber. Viability of cells, optical transparency of membranes and strong adhesion of PLLA to PDMS were observed, thus confirming the suitability of the prepared membranes for use in organs-on-a-chip devices.