RNA interference (RNAi) has become a powerful and commonly used genetic tool for studying gene function. Vector-based short hairpin RNAs (shRNAs) trigger long-lasting RNAi effects compared to chemically synthesized siRNAs especially when they are imbedded in a lentiviral vector. In this chapter, a cost-effective method for shRNA design and expression is described. The strategy uses a carefully selected shRNA loop sequence and an antisense-loop-sense stem structure. Single and multiple shRNAs were expressed from the same vector, and thus the current design strategy has a great application potential in genomics.