RNA interference (RNAi) library was pioneered in C. elegans in a broad range of organism-based screens. During the past few years, RNAi has become a powerful tool to silence the expression of genes and analyze their loss-of-function phenotype in mammalian cells. There are two types of RNAi library, synthetic oligonucleotide library and vector library, and two screening strategies, high throughput and selective screenings. Short hairpin RNA (shRNA), which is processed intracellularly into short duplex RNAs and has siRNA-like properties, can mediate persistent gene silencing after stable integration of the vector into the host-cell genome. In combination with the shRNA vector library and suitable screening strategies, much greater depth will be added to the functional understanding of therapeutic applications of potential targets in oncology.