Natural dissolved organic matter (DOM) in aquatic systems plays many environmental roles: providing building blocks and energy for aquatic biota, acting as a sunscreen in surface water, and interacting with anthropogenic compounds to affect their ultimate fate in the environment. Such interactions are a function of DOM composition, which is difficult to ascertain due to its heterogeneity and the co-occurring matrix effects in most aquatic samples. This review focuses on current approaches to the chemical structural characterization of DOM, ranging from those applicable to bulk samples and in situ analyses (UV-visible spectrophotometry and fluorescence spectroscopy) through the concentration/isolation of DOM followed by the application of one or more analytical techniques, to the detailed separation and analysis of individual compounds or compound classes. Also provided is a brief overview of the main techniques used to characterize isolated DOM: mass spectrometry (MS), nuclear magnetic resonance mass spectrometry (NMR) and Fourier transform infrared spectroscopy (FTIR).