Advanced dynamic anaerobic digestion models, such as ADM1, requires both detailed organic matter characterisation and intimate knowledge of the involved metabolic pathways. In the current study, a methodology for municipal sludge characterization is investigated to describe two key parameters: biodegradability and bioaccessibility of organic matter. The methodology is based on coupling sequential chemical extractions with 3D fluorescence spectroscopy. The use of increasingly strong solvents reveals different levels of organic matter accessibility and the spectroscopy measurement leads to a detailed characterisation of the organic matter. The results obtained from testing 52 municipal sludge samples (primary, secondary, digested and thermally treated) show a successful correlation with sludge biodegradability and bioaccessibility. The two parameters, traditionally obtained through the biochemical methane potential (BMP) lab tests, are now obtain in only 5 days compared to the 30 to 60 days usually required. Experimental data, obtained from two different laboratory scale reactors, were used to validate the model. The proposed approach showed a strong application potential for reactor design and advanced control of anaerobic digestion processes.