Mass spectrometry has been extensively used in the identification of protein and its posttranslational modification (PTM). Mass spectrometry coupled with nano high performance liquid chromatography (HPLC) could improve its resolution and efficiency. Ubiquitination, one kind of the important protein posttranslational modifications (PTM), plays a pivotal role in dynamic balance and functional regulation of protein. A new strategy taking the advantage of immunoprecipitation, 2D nano HPLC and matrix-assisted laser desorption/ionization-time of flight mass spectrometry was established to identify the ubiquitination of endogenous protein in mammalian cells. By using this strategy, the ubiquitinated sites of c-ABL in K562 leukemia cells was successfully identified. Thus, it may provide a valuable tool to characterizing the ubiquitination of endogenous proteins under physiologic and pathologic conditions.