The incorporation of nanofluidic elements between microfluidic channels to form hybrid microfludic/nanofluidic architectures allows the extension of microfluidic systems into the third dimension, thus removing the constraints imposed by planarity. Measuring and understanding the behavior of these devices creates new analytical challenges due to the inherently small volumes, short length scales, small numbers of analyte molecules, and new physical phenomena involved. To this end, robust and reproducible methods were developed to fabricate hybrid microfluidic/nanofluidic systems where single, high aspect ratio polymer nanopores (100 nm < d < 500 nm) provide fluidic connection between vertically separated microfluidic channels. The current-voltage characteristics of these devices were investigated to determine both the internal dimensions of the FIB-milled nanopores used in their fabrication as well as to shed light on fundamental fluidic behavior arising from the coupling of the microfluidic and nanofluidic elements in the integrated system. A custom axially opposed dual confocal fluorescence microscope was constructed to study analyte transport in these hybrid microfluidic/nanofluidic devices. The behavior of analyte molecules ranging from small molecules ( i.e. fluorescein) to macromolecules (i.e. DNA) during electrokinetic transport in these systems was studied using both this confocal fluorescence microscope as well as wide field fluorescence microscopy to elucidate relationships between nanopore surface charge, analyte charge, analyte size, and applied electrical potential. The inherent optical characteristics of the devices were shown to limit the ultimate spatial resolution and sensitivity of the measurement system, but single file transport of fluorescently labeled DNA molecules was observed.