Basidiomycetes inhabiting lignocellulose comprise an important class of filamentous fungi with ecological and biotechnological relevance. Extracellular enzymes of wood-degrading fungi such as laccases, manganese-dependent (or independent) peroxidases, cellulases and xylanases are of considerable interest for biotechnological applications. Still, proteomic studies of fungal secretomes based on 2-DE can be very challenging due to low protein concentrations and variable amounts of fungal metabolites. Comparison of different standard methods for protein precipitation has demonstrated their limited applicability to fungal secretomes. The extracellular metabolites impair standard methods for protein quantification and can result in a strong overestimation of total protein. We have developed an optimized protocol for the precipitation of extracellular proteins from liquid cultures of Coprinopsis cinerea growing in an exponential phase on a complex media. We found that a considerable amount of gelatinous material can be removed from the liquid culture supernatants by high-speed centrifugation. Fungal proteins can be effectively enriched by TCA precipitation and coprecipitated metabolites hampering 2-DE can be removed through the application of Tris/acetone. Following our protocol makes it possible to concentrate proteins from culture supernatants and to simultaneously remove most of the impeding compounds from a given sample. We have applied this procedure in the 2-DE of secretomes from the model organism C. cinerea as well as other basidiomycetes such as Pleurotus ostreatus, Phanerochaete chrysosporium, Polyporus brumalis and Schizophyllum commune.