Although the identification of ubiquitin-conjugated lysine residues on target proteins is extremely significant, to date it is generally quite difficult to identify ubiquitinated sites by usual mutation analysis. More recently, the technology of mass spectrometry is answering these difficult questions. In this chapter, we introduce the method of purification of ubiquitinated target proteins using affinity chromatography with anti-polyubiquitin antibody and the identification of ubiquitinated lysine residues on target proteins using mass spectrometry. Using these techniques, we can obtain comprehensive information about ubiquitinated proteins in various cells and tissues.