Aquatic humic substances (AHSs) are major constituents of dissolved organic matter (DOM) in freshwater, where they perform a number of important ecological and geochemical functions, yet no method exists for quantifying all AHSs. We have developed a method for the quantitative analysis of AHSs based on their carbon concentration. Our approach includes: (1) the development of techniques for clear-water samples with low AHS concentrations, which normally complicate quantification; (2) avoiding carbon contamination in the laboratory; and (3) optimizing the AHS adsorption conditions.