RNA interference (RNAi) has modernized the studies to determine the role of a particular/novel gene. RNAi is a biological process where RNA molecule inhibits the expression of a particular gene by targeting and destructing specific mRNA molecules. RNAi is also known as posttranscriptional gene silencing (PTGS), co-suppression, and quelling. Silencing of target genes by RNAi technology came into the limelight just after discovery of plant defense mechanism against virus, where it was believed that plant encode short, noncoding region of viral RNA sequences, which after infection recognizes and degrades viral mRNA. These short and noncoding RNA sequences might be against viral DNA/RNA polymerase and other important genes necessary for viral infection and multiplication. On the theme of the above concept of plant virologist introduced short nucleotides sequence into the viruses and expression of target genes in the infected plants was found to be suppressed. This most popular phenomenon is known as “virus-induced gene silencing” (VIGS) and brings the boom in the era of biotechnologists. This chapter is detailed with the mechanism and recent advancement of RNAi technology.