The study of organic matter in wastewater is a major regulatory and environmental issue and requires new developments to identify non-biodegradable refractory compounds, produced mainly by thermal treatments. Recent advances linking physicochemical properties to spectroscopic analyzes (UV, Fluorescence, IR) have shown that the refractory property is favored by several physicochemical parameters: weight, hydrophobicity, aromaticity and chemical functions. Currently, the most effective developments for the quantification of refractory compounds are obtained with hyphenated methods, based on steric separation of the macromolecular species by steric exclusion chromatography (SEC)/PDA/Fluorescence systems. Hyphenated techniques using High Resolution Mass Spectrometry (HRMS), ultra-high-resolution mass spectrometry with Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and NMR have been developed to analyze macromolecules in wastewater with minor sample preparation procedures. A particular class has been identified, the melanoidins, generated by Maillard reactions between sugars, amino acids, peptides and proteins present in wastewater and sludge, but low molecular weight compounds formed as intermediates, such as ketones, aldehydes, pyrazines, pyridines or furans, are also recalcitrant and are complex to identify in the complex matrices. The lack of available standards for the study of these compounds requires the use of specific techniques and data processing. Advances in chemometrics are obtained in the development of molecular or physicochemical indices resulting from the data generated by the analytical detectors, such as aromaticity calculated by SUVA254 and determined by UV, fluorescence, molar mass, H/C ratio or structural studies (measuring the amount of unsaturated carbon) given by hyphenated techniques with SEC. It is clear that nitrogen compounds are widely involved in refractoriness. New trends in nitrogen containing compounds characterization follow two axes: through SEC/PDA/Fluorescence and HRMS/NMR techniques with or without separation. Other techniques widely used in food or marine science are also being imported to this study, as it can be seen in the use of “omics” methods, high-performance thin layer chromatography (HPTLC) and chromatography at the critical condition, rounding out the important developments around SEC. While improving the performance of stationary phases is one of the challenges, it results in a fundamental understanding of the retention mechanisms that today provide us with more information on the structures identified. The main objective of this review is to present the spectroscopic and physicochemical techniques used to qualify and characterize refractoriness with a specific focus on chemometric approaches.