Field-flow fractionation (FFF) is one of the promising alternative separation techniques to size exclusion chromatography (SEC). In this study, water soluble polymers including Bovine Serum Albumin (BSA), pullulan, poly(ethylene oxide) (PEO), sodium poly(styrene sulfonate) (NaPSS), and partially saponified poly(vinyl alcohol) (PVA) have successfully been characterized by a means of the FFF technique, fitted with a multiangle laser light scattering (MALS) detector in 0.1 M Na NO3 as an eluent at 25 degrees C. The factors which affect the retention time, t(r) and the separation factor, R-s of the monomer and dimer of BSA are the thickness of spacer, w, channel flow rate, V-CHF, cross-flow rate, V-CRF, focusing time, injection volume, and polymer concentration, C-p. We found that the retention time, t(r) in the FFF follows the equation of t(r)similar to[V-CHF](-1.0)[V-CRF](1.0)[w](2.0) in agreement with theory. Several cross-flow rates were examined for the characterization of the water soluble flexible polymers and the polyelectrolyte, and an appropriate gradient method for these polymers is found to be described by the equation of V-CRF = A exp (-Bt(r)), where A and B are constant. For the polyelectrolyte, the retention time significantly varies with salt concentration, due to the balance between change in the conformation of polyelectrolyte chains and the electrostatic exclusion from the membrane filter.