Conjugation of SUMO to substrates is involved in a large number of cellular processes. Typically, SUMO is conjugated to lysine residues within a SUMO consensus site; however, an increasing number of proteins are sumoylated on non-consensus sites. To appreciate the functional consequences of sumoylation, the identification of SUMO attachment sites is of critical importance. Discovery of SUMO acceptor sites is usually performed by a laborious mutagenesis approach or using mass spectrometry (MS). In MS, identification of SUMO acceptor sites in higher eukaryotes is hampered by the large tryptic fragments of SUMO1 and SUMO2/3. MS-search engines in combination with known databases lack the possibility to search MSMS spectra for larger modifications, such as sumoylation. Therefore we developed a, simple and straightforward database search tool (“ChopNSpice”) that successfully allows identification of SUMO acceptor sites from proteins sumoylated in vivo and in vitro. By applying this approach we have identified SUMO acceptor sites in amongst others endogenous SUMO1, SUMO2, RanBP2 and Ubc9.