Polyubiquitinated proteins tagged with multi-ubiquitin chains are substrates preferred by the 26 S proteasome (a ubiquitin/ATP-dependent proteolytic complex). Here, we developed a simple method for the efficient preparation of polyubiquitinated proteins which are degraded by the 26 S proteasome in an ATP-dependent manner. Our efficient method enabled us to produce ten monoclonal antibodies that recognized the multi-ubiquitin chains of the polyubiquitinated proteins, but not free ubiquitin or the protein moieties. Eight of the antibodies recognized only the multi-ubiquitin chains of the polyubiquitinated proteins, while the other two antibodies cross-reacted with mono-ubiquitin and methyl-ubiquitin, both of which are linked to proteins via an isopeptide bond, as well as with the multi-ubiquitin chains. Thus these antibodies are novel and useful tools for the identification and quantification of polyubiquitinated proteins in various cells and tissues under physiological and pathological conditions.