This Ph.D. dissertation describes the main project of my academic research at the University of Chicago as a graduate student: the identification of substrates of ubiquitin E3 ligases using our lab’s unique system called orthogonal ubiquitin transfer (OUT). Each chapter describes different, sequential steps in how the goal was finally achieved; each chapter builds on the previous, akin to rungs in a ladder, and together they demonstrate a logical, sequential way of approaching and solving the problem in distinct steps. Chapter 1 describes background information about the ubiquitin-proteasome system, as well as the Yin lab’s previous research and established information on OUT at the time of my joining the program, and also includes some parts of my work in the earliest year of my study. Chapter 2 describes my own part in the development of a ubiquitin ligase for OUT via rational design and specific point mutagenesis, as well as my work and analysis on the E2-E3 interface. Chapter 3 involves the construction of a phage display library displaying the U-box domain of the ubiquitin ligase E4B, as well as the screening for active mutants, and the analysis of the consensus of the sequences of the mutants selected. Chapter 4 involves the characterization of the selected mutants and the assaying of their activity with OUT, as well the application of the selected amino acid sequences onto corresponding residues on other U-box E3 ligases, namely, the yeast homolog Ufd2 and CHIP, and the activity of those mutant analogs on putative substrates. Chapter 5, the last chapter, describes the practical application of the whole OUT cascade in vivo, and the proteomics screening for substrates, and the verification of such substrates.