Updated project metadata. Metaproteomic studies of the rumen microbiota are challenged by the need of optimized sample preparation protocols in order to retrieve an enhanced amount of prokaryotic instead of plant and bovine derived cells before protein extraction and subsequent LC-MS/MS analysis. The present study evaluates three different protocols applied to the rumen microbiota either attached to plant fibres or present as planktonic cells. The findings of our work suggest the integration of cheesecloth-gauze filtration in sample preparation to achieve a better protein identification ratio.