Nanofluidic devices are structures having at least one dimension in the submicron range, which is of the same order of magnitude as the sizes of biomolecules and bioparticles such as proteins and viruses. As a result, size-selective separations are important applications for nanofluidics. Well-defined micro or nano device structures fabricated via micromachining have greatly reduced sample consumption and enabled separations in a parallel fashion, promising significant speed and resolution advantages over conventional size separation techniques, such as gel electrophoresis and size exclusion chromatography. In collaboration with others, I have developed a size separation method using nanofluidic devices consisting of an array of parallel planar nanochannels with varying heights. Separation of nanoparticles is accomplished by simply flowing a liquid suspension of the particles through the nanochannels via capillary action. When a mixture of particles arrives at an interface, where the channel steps from a deeper to a shallower segment, larger particles become trapped and smaller particles pass through, thereby achieving separation. In this dissertation, I demonstrated the successful trapping of polymer nanobeads and two types of virus capsids (30 nm hepatitis B virus capsids and 120 nm herpes simplex virus type 1 capsids) using nanochannels with two different channel height segments. Furthermore, I studied the fractionation of nanoparticles in nanochannels with three different channel height segments. The effects of surfactants and an alternating current electric field on particle distribution were investigated, both of which aided in the prevention of channel clogging. Most recently, I applied the nanosieving method for separating lipoproteins, which are important in the diagnosis of cardiovascular disease. Promising results were obtained, indicating that the major lipoprotein classes, including intermediate density lipoproteins (IDL, 23-35 nm), low-density-lipoproteins (LDL, 18-25 nm) and high-density-lipoproteins (HDL, 5-12 nm), may eventually be fractionated using three-segment nanochannels. To successfully fractionate lipoprotein mixtures, characterization of flow dynamics in three-segment nanochannels, passivation of the surface to prevent nonspecific protein adsorption, application of an electric field to help particles overcome an energy barrier, and use of multi-color fluorescent labeling to assist detection are required. I studied the channel passivation performance of polyethylene glycol (PEG) and used dual-color fluorescence detection for the separation of a binary protein mixture. Finally, I fabricated channels with monotonically changing barrier heights and demonstrated differential trapping of polymer beads. The data trend followed a slit model derived from a model developed by Giddings for size exclusion chromatography.