Leafhoppers pose a severe threat to cotton crops, causing substantial yield losses. The use of synthetic insecticides for pest control has drawbacks such as resistance development, environmental harm, and health risks. In this context, RNA interference (RNAi)-based strategies have emerged as an alternative for leafhopper management. RNAi involves introducing double-stranded RNA molecules (dsRNAs) that target vital genes, disrupting their function and causing reduced fitness or death. Specific target genes are identified and corresponding dsRNA molecules are produced and delivered to leafhoppers through various methods. This chapter presents a case study of Amrasca biguttula, showcasing the potential of RNAi in cotton leafhopper control. Despite delivery and cost challenges, RNAi offers precise, safe, and eco-friendly benefits, making it a valuable tool for managing this significant cotton pest. The chapter outlines techniques for mass rearing leafhoppers, gene selection, RNA isolation, cDNA synthesis, primer design, PCR amplification, and in vitro transcription for dsRNA synthesis. It also includes a protocol for RNAi diet bioassay for bio-efficacy testing and molecular characterization of dsRNA-treated insects. Proper handling and use of RNase-free reagents are emphasized throughout these processes. Overall, we present an RNAi protocol as a valuable tool for controlling leafhopper infestations in cotton research experiments.