Ubiquitin and polyubiquitin chains target proteins for a wide variety of cellular processes. Ubiquitin-mediated targeting is regulated by the lysine through which the ubiquitins are linked as well as the broader ubiquitin landscape on the protein. The mechanisms of this regulation are not fully understood. For example, the canonical proteasome targeting signal is a lysine 48-linked polyubiquitin chain, and the canonical endocytosis signal is a lysine 63-linked polyubiquitin chain. However, lysine 63-linked polyubiquitin chains can also target substrates for degradation. Biochemical studies of ubiquitinated proteins have been limited by the difficulty of building proteins with well-defined polyubiquitin chains. Here we describe an efficient and versatile method for synthesizing ubiquitin chains of defined linkage and length. The synthesized ubiquitin chains are then attached to any protein containing a ubiquitin moiety. These proteins can be used to study ubiquitin targeting in in vitro assays in the tightly controlled manner required for biochemical studies.