Flow field-flow fractionation (FlFFF) is commonly used to determine the molecular weight (MW) of water-soluble polymers. Calibration is usually achieved using monodisperse MW standards, which restricts the determination of absolute MW distribution to a few commonly used polymers. To overcome this limitation, a calibration method using polydisperse standards, which was first developed for thermal field-flow fractionation, has been modified for use in FlFFF. The method was tested using a series of water-soluble polymer samples with known average MW values. The calibration method using polydisperse standards has been validated, and the method should expand the sample range for FlFFF analysis.