The application of the fluorescence excitation-emission matrix (EEM) approach and liquid chromatography–organic carbon detection (LC–OCD) analysis for the characterization of hydraulically reversible and irreversible fouling species, extracted from hollow fiber ultrafiltration (UF) membranes used in drinking water treatment, was demonstrated. Hydraulically reversible and irreversible fouling species were extracted from two pilot UF membrane systems operated in parallel with lake water as the feed. Two membrane cleaning protocols, hydraulic- and chemical-based (NaOCl and citric acid) cleaning, were considered. Colloidal/particulate matter together with protein-like and metal species in water appeared to contribute to the formation of a hydraulically removable fouling layer on the membranes. Hydraulically irreversible fouling, in contrast, was impacted considerably by humic substances (HS) and protein-like matter. The formation of an irreversible fouling layer was also likely influenced by interactions between the colloidal/particulate matter and metal species together with HS and protein-like matter. LC–OCD analysis revealed the presence of predominant levels of lower molecular weight HS-like matter – compared to the HS-like matter commonly present in lake water – in the citric acid extracted foulant fraction. The permeability loss due to hydraulically irreversible UF fouling was considerably greater than the permeability loss due to hydraulically reversible UF fouling. A permanent permeability loss (∼25–35% of the initial permeability) was present even after the application of considerably strong chemical cleaning protocols on both pilot systems. This study indicated that the fluorescence EEM approach can be applied for monitoring and characterization of membrane cleaning procedures and as a potential diagnostic tool for assessing the effectiveness of hydraulic- and chemical-based cleaning protocols employed in UF drinking water treatment operations using rapid off-line measurements. On the other hand, since the LC–OCD analysis technique is a comparatively time consuming method, it may be used for verification of the fluorescence EEM-based results of the foulant fractions.