Ubiquitin‐mediated proteolysis is a major pathway of protein degradation that regulates
numerous cellular processes. An understanding of the circumstances that contribute to the
ubiquitylation of a specific protein can yield vast insight into its regulation. This article
examines multiple procedures that explain whether a protein is ubiquitylated and suggests
methods to investigate the factors that specifically target the substrate for ubiquitylation, as
well as the site of ubiquitin conjugation.