A multi-method extraction protocol based on mechanical, ionic and hydrophobic methods was investigated on two types of activated sludge samples. Extraction methods were chosen with regards to optimal protein yield without cell disruption. Sonication, EDTA and Tween extraction methods were selected and combined. The total amount of protein released by the multi-method protocol sums up to 191 and 264 mg equiv. BSA/g VSS for the two different sludge samples. Protocol repetition on the same sample showed that protein yield after each successive protocol fitted an exponential curve model. The total amount of extractable proteins was evaluated by model predictions, 423 and 516 mg equiv. BSA/g VSS for the two sludge samples. The multi-method extraction protocol appears relevant for harvesting a representative quantity of proteins from the original sample (45-49%), moreover the multi-method criterion of the protocol also offers a heterogeneous pool of proteins. Thus, further qualitative studies may not be biased by the extraction protocol. (C) 2008 Elsevier Ltd. All rights reserved.