An overview of different characterization methods and the possibilities for describing structural elements of humic substances from aqueous environments (HS, or refractory organic substances (ROS)) is given within this review. The application of different analytical methods for the qualitative and quantitative characterization of HS, including physical/chemical analysis (elemental analysis, acid/base titration), spectroscopic methods (UV/VIS, NMR, fluorescence, mass spectrometry), fractionation methods (gel chromatography, flow-fieldflow-fractionation), and degradation methods (oxidation, Aquatic Sciences pyrolysis, hydrolysis) are discussed. The paper focuses on the most commonly used chemical, spectroscopic and chromatographic methods used in the last few years. Aspects concerning information related to newer procedures are considered. Case studies representing results gained from reference samples isolated from brown water and wastewater effluents are given to cross-check results gained from several methods. The influence of different isolation procedures on the specific character of the fractions is also discussed.