An important step in the ubiquitin proteolytic cascade is specific recognition of the substrate by a member of the ubiquitin ligases family of proteins—an E3, that is followed by generation of the polyubiquitin degradation signal. For most substrates, it is believed, though it has been demonstrated experimentally only for a few, that the first ubiquitin moiety is conjugated, via its C-terminal Gly76 residue, to an ε-NH2 group of an internal Lys residue. Recent findings indicate that for several proteins, the first ubiquitin moiety is fused linearly to the α-NH2 group of the N-terminal residue. Important biological questions relate (1) to the evolutionary requirement for an alternative mode of ubiquitination, (2) to the identity of the set of proteins in the proteome that undergoes N-terminal ubiquitination, and (3) to the relationship between N-terminal ubiquitination and N-terminal acetylation. In this chapter we describe methods that will enable researchers to identify this novel mode of ubiquitination.