Glycosylphosphatidylinositol (GPI) is used for anchoring many cell surface proteins to the plasma membrane. Biosynthesis of GPI anchor, its attachment to proteins, and modification of GPI-anchored proteins (GPI-APs) en route to the plasma membrane are complex processes (Ferguson, 1999; Kinoshita and Inoue, 2000). GPI-AP-defective mutant cell lines derived from CHO and other cells have been very useful in elucidating GPI biosynthetic pathway and cloning genes involved in these processes. In this chapter, we overview GPI-AP biosynthesis, establishment and characterization of GPI-AP-defective mutant cell lines, expression cloning using those mutant cells, and characteristics of GPI-AP-defective mutant cell lines.