Microfluidic devices with porous membranes for molecular sieving, metering, and separation of analyte fluids. In one aspect, a microfluidic device includes a substrate having input and output microfluidic channel sections separated by a porous membrane formed integral to the substrate. In another aspect, the porous membrane may comprise a thin membrane that is sandwiched between upper and lower substrate members. The microfluidic device may include one or a plurality of porous membranes. In one embodiment, a plurality of porous membranes having increasingly smaller pores are disposed along portions of a microfluidic channel. In another embodiment, a cascading series of upper and lower channels are employed, wherein each upper/lower channel interface is separated by a respective porous membrane. In another aspect, a porous membrane is rotatably coupled to a substrate within a microfluidic channel via a MEMS actuator to enable the porous membrane to be positioned in filtering and pass-through positions.