Construction of expression vectors is imperative for many areas of biological research and the biotechnology industry. Modular cloning systems for expression vector construction offer a labor- and cost-effective alternative to overcome drawbacks associated with traditional cloning methods. We developed an Extensible Mammalian Modular Assembly toolkit (EMMA) as an efficient and versatile tool to facilitate the construction of functionally diverse mammalian expression vectors from a standardized library of DNA parts. This system supports both hierarchical and combinatorial assembly, and is adaptable for automation. In this chapter, we describe the protocols to construct libraries of DNA parts and assemble these parts into mammalian expression vectors using EMMA.