This paper introduces a novel natural organic matter (NOM) fractionation technique using solid-phase extraction cartridges. The new technique requires only 6 hours of fractionation time, which is much faster than traditional fractionation techniques (24 hours). It uses three Bond Elute ENV cartridges (Varian, Inc., Lake Forest, California), one Strata X-C cartridge (Phenomenex, Torrance, California), and one Strata X-AW cartridge (Phenomenex) in series and was tested by using to fractionate NOM from Suwannee River, Georgia (SRNOM) and Red River, Minnesota (RRNOM). Hydrophobic acid was a major fraction and accounted for 66 to 70% and 36% of SRNOM and RRNOM, respectively. The NOM fractions obtained from the developed method were characterized using Fourier transformed infrared spectroscopy and 13C nuclear magnetic resonance. The acid fractions of SRNOM mainly consisted of carboxylic acids. An application of this new technique was demonstrated by using it to investigate the effectiveness of water treatment processes in removing different NOM fractions.