The feasibility of metaproteomic analysis of the microbial communities underpinning anaerobic wastewater treatment relies on efficient protein extraction and separation techniques. The microorganisms involved in anaerobic digestion (AD) of wastewater typically aggregate to form tightly organised, spherical granules, from which protein extraction is challenging. Here, we compare two methods of protein extraction, one using a French press previously used successfully to analyse the proteome of an activated sludge [Wilmes, P., Bond, P. L., Environ. Microbiol. 2004, 6, 911-920] and one using sonication developed in the context of pure culture [Abram, F., Wan-Lin, S., Wiedmann, M, Boor, K J., Coote, P., Botting, C., Karatzas, K. A. G., O'Byrne, C. P, Appl. Environ. Microbiol. 2008, 74, 594-604.]. We used 2-DE to carry out this comparison. The protein extraction using the sonication method resulted in a significant increase in the number of protein spots and higher quality 2-D gels.