A method to fabricate biocompatible, polymer microsystems with integrated electrical and fluidic functionality is presented. The process flow utilizes laser ablation, microstenciling, and heat staking as the techniques to realize multi-layer microsystems with microchannels, thru and embedded fluidic/electrical vias, and metallic electrodes/contact pads. A six-layer multi-functional cellular analysis system is demonstrated as a test vehicle for the fabrication technology. The analysis system contains fluidic microchannel/via networks for cell positioning and chemical delivery as well as electrodes for electrophysiological studies. The microsystem is constructed out of multiple layers of 50.8 μm thick sheets of Kapton ® (DuPont). Kapton ® provides a biocompatible substrate that is flexible while maintaining structural stability, and it adds the ability to operate in high temperature and other harsh environments. Kapton ® also lends itself well to laser ablation and multi-layer bonding. Microchannels with widths of 400 μm as well as thru-hole fluidic vias with minimum diameters of 4 μm (aspect ratios of over 12:1) are laser ablated through the polyimide sheets using an excimer laser and a CO 2 laser. Electrical traces and contact pads with minimum feature sizes of 10 μm are patterned onto the flexible polyimide sheets using microstenciling. Microstenciling enables the metal patterning to be performed repeatedly without having to use photolithography on any polymer sheet. The patterned layers are bonded using heat staking at a temperature of 350 °C and a pressure of 1.65 MPa for 60 min. Tests show that the multiple layers of the microsystem are bonded adequately and that the fluidic channel can withstand the pressure resulting from a flow forced through the channel at flow rates within the range of interest for this study (0.2–1.4 mL/h) without delaminating or stretching. This multi-layer technology can be used to create microfluidic devices for many application areas requiring biocompatibility, relatively high temperature operation, or a flexible substrate material.