The identification of protein modification sites is an important step toward understanding the biological role of covalent modifications. For example, the mapping of phosphorylation sites and analyses of phosphorylation site mutants have tremendously contributed to our knowledge of different cellular processes. Given the diverse functions of ubiquitination, similar studies with ubiquitin attachment site mutants are becoming increasingly important in understanding the molecular roles of ubiquitination. Relatively few studies to date have mapped ubiquitination sites, and in almost all cases the identification of the acceptor lysines were based on indirect evidence (Petroski and Deshaies, 2003; Scherer et al., 1995); that is, mutation of particular lysines to arginines blocked ubiquitination. Direct evidence for ubiquitin attachment sites has been obtained by mapping of hydroxylamine-derived peptides from ubiquitinated proteins (Chau et al., 1989); however, these experiments can be very challenging. Recent advances in protein mass spectrometry have enabled ubiquitinated lysine residues to be identified directly, thereby providing more convincing evidence for the exact location of the modification (Flick et al., 2004; Peng et al., 2003). In addition to mapping attachment sites, mass spectrometry can also be used to determine the type of ubiquitin chain linkage (Flick et al., 2004; Peng et al., 2003). In vivo evidence for the covalent attachment of the carboxyl terminus of one ubiquitin molecule to lysine residues in several locations in a different ubiquitin molecule demonstrates the complexity of ubiquitin biology (Peng et al., 2003). These different ubiquitin chain topologies can dramatically affect the molecular function of ubiquitin chains (Hoege et al., 2002; Spence et al., 1995), and, hence, the mass spectrometric determination of the ubiquitin chain architecture can provide important insight into the mechanisms of ubiquitin function. This chapter describes mass spectrometric approaches for identifying ubiquitin acceptor lysines on target proteins and analyzing the ubiquitin chain topology.