Despite the importance of anaerobic sludge extracellular polymeric substances (EPSs), their characterization is limited to information regarding their chemical classes and molecular size. This work explores the possibility of using proteomic techniques to study the proteins present in this matrix. Thus, this paper compares eight EPS extraction methods regarding extraction yield, protein/carbohydrate ratio, size distribution profile and suitability to sodium dodecyl sulfate–polyacrylamide gel electrophoresis analyses. Despite the differences found in quantification and size exclusion chromatography assays, the band profile found for all methods was very similar. Considering the band pattern, extraction time and background level, heating method followed by ammonium sulfate precipitation proved to be the most appropriate method for gel-based analyses of anaerobic sludge EPS proteins.