This present study involves the assessment of activated sludge based on the roles of proteins and extracellular polymeric substances (EPS) in continuous flow activated sludge treatment systems. The first objective of the study is to investigate the applicability of total protein quantification to estimate the quantity of active biomass. Activated sludge samples from three industrial wastewater treatment plants (WWTPs) and a lab-scale plant were analysed for this purpose. The total protein concentration of the sludge was compared to the conventional biomass parameters, i.e. suspended solids (SS) and volatile suspended solids (VSS), and correlated to the respirometric activity measured by the oxygen uptake rate (OUR). In both industrial and lab-scale plant samples, VSS showed the strongest correlation to the OUR, followed by SS, while total protein concentration showed weak or no correlation. Therefore, it is concluded that there was no direct relationship between protein content in activated sludge to the activity thus total protein concentration is not suitable to be used to measure active biomass. Most problems related to activated sludge systems involve sludge separation problems. The second objective of this study was to determine the roles of the EPS components in sludge settleability and dewaterability. For this purpose, the protein, humic acid and carbohydrate contents of bound EPS were correlated to the sludge volume index (SVI) and capillary suction time (CST), which represented sludge settleability and dewaterability respectively. The results were contradictory between the industrial and lab-scale plant samples and the correlations, if any, were weak. As the EPS properties seemed to differ from plant to plant, defining the roles of the EPS from their ‘bulk’ contents was found to be inconclusive and speculative at best. The third aim was to investigate the total protein and EPS at a molecular level to obtain more conclusive information on how they affect sludge properties and nutrient removal efficiency. For this purpose, activated sludge samples were subjected to nitrogen and phosphorous limiting conditions, two common problems in activated sludge process. Label-free quantification (LFQ) proteomics analyses were performed to determine the qualitative and quantitative changes in the protein expressions of total protein and EPS under these conditions. Differential protein expression illustrated that the system adopted different strategies towards mitigating the effects of nutrient deficiency. N-limiting period caused the upregulation of various antioxidant enzymes and DNA-binding protein from starved cells (Dps), signifying the system reacted to protect the cells from oxidative stress and DNA damage. The system also reacted by increasing nitrogen uptake through GS/GO-GAT system and the expressions of urease and nitrogenases. Phosphorous limitation also caused the upregulation of various antioxidant enzymes but it was not clear how the system regulated phosphorous uptake in this condition. With the help of proteomics, there is now more appreciation for the roles of the EPS in activated sludge process. EPS was found to contain chaperones, transport proteins, antioxidant enzymes, and other proteins that are essential for cell integrity under normal conditions as well as under environmental stress. This study proved that the application of quantitative proteomics using LFQ-MS is beneficial in retrieving various information about the state of sludge and gaining further understanding of the activated sludge process.