Currently, RNA interference technology is one of the most powerful tools in molecular biology and has been widely used in genetic manipulation. In addition to chemically synthesized small interfering RNA (siRNA), vector-based methods have been developed for stable gene silencing by the expression of a single short-hairpin RNA (shRNA). The artificially expressed RNA molecules are processed to form a silencing complex that causes the specific degradation of its target mRNA. However, silencing vectors containing a single shRNA-expressing sequence sometimes induce only poor knockdown. In order to improve the knockdown efficiency using shRNA, the multiple shRNA-expressing sequences were introduced into a single plasmid vector. Compared with the conventional single shRNA-expression vector, the multiple shRNA-expression vectors confer higher yields of stable clones with efficient knockdown and better correlations between knockdown level and the expression level of second marker gene, enhanced green fluorescent protein, in the vector. These features are very helpful for establishing stable knockdown clones and the detailed procedure is described in this chapter.