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Asymmetrical flow field-flow fractionation (AF4) efficiently separates various macromolecules and nano-components of natural waters according to their hydrodynamic sizes. The online coupling of AF4 with fluorescence (Fluo) and UV absorbance (UV) detectors (FluoD and UVD, respectively) and inductively coupled plasma–mass spectrometry (ICP-MS) provides multidimensional information. This makes it a powerful tool to characterize and quantify the size distributions of organic and inorganic nano-sized components and their interaction with trace metals. In this study, we developed a method combining thiol labeling by monobromo(trimethylammonio)bimane bromide (qBBr) with AF4–FluoD to determine the size distribution and the quantities of thiols in the macromolecular dissolved organic matter (DOM) present in highly colored DOM-rich water sampled from Shuya River and Lake Onego, Russia. We found that the qBBr-labeled components of DOM (qB-DOM) were of humic type, characterized by a low hydrodynamic size (dh