The number and complexity of natural organic matter (NOM) species limits identification of individual NOM compounds. The objective of this study was to employ several characterization techniques (resin fractionation, high performance size exclusion chromatography (HPSEC), and strategic UV254 absorbance) to samples from seven surface water sites in North America, and overcome the shortfalls of each tool. Resin fractionation indicated the samples were all high in hydrophobic acids (HOA), hydrophilic neutrals (HIN) and hydrophilic acids (HIA). Site B was the only site where HIAs were the highest NOM contributors. In the HPSEC analysis, each fraction exhibited a particular molecular weight (MW) range: 100–300 Da (HIN), 1-2 kDa (HOA), and the HIA fractions exhibited MWs between these two ranges. Strategic UV254 measurements were taken at two sites to supplement the HPSEC results, and determine the difference in UV absorbance per unit dissolved organic carbon (SUVA value). Most fractions showed SUVA values of approximately 5 L/mg-m; however, the hydrophilic bases and hydrophobic neutral fractions could not be accurately evaluated due to the very low DOC concentrations for these two fractions (< 0.2 mg/L). These methods are complimentary NOM characterization techniques, and the combined methodology addresses the analytical limits of each tool.