RNA interference (RNAi) is a promising new class of pharmaceutical drugs that can specifically and potently inhibit disease targets. Traditional pharmaceutical approaches face challenges in identifying highly specific and potent drug candidates, especially for intractable targets. RNAi offers a powerful method for rapidly identifying inhibitors of disease targets across all molecular classes. Numerous proof-of-concept studies in animal models demonstrate the broad potential of RNAi therapeutics. The major challenge in drug development is identifying effective delivery strategies that can be translated to the clinic. Advances in this area and the initiation of clinical trials with RNAi therapeutic candidates suggest a potential transformation in modern medicine. RNAi is a fundamental cellular mechanism for silencing gene expression that can be harnessed for drug development. It can be used to reduce the expression of pathological proteins, including those difficult to modulate with traditional pharmaceutical approaches. RNAi therapeutics have the potential to significantly impact modern medicine. The mechanism involves the enzymatic cleavage of target mRNA, leading to decreased protein abundance. Synthetic siRNAs leverage the naturally occurring RNAi process, while viral delivery of shRNAs is an alternative strategy. This review provides an overview of the molecular mechanism of RNAi, the in silico design of siRNAs and shRNAs, chemical modifications that enhance stability, and strategies for cellular delivery. It summarizes the numerous publications demonstrating the robust efficacy of RNAi in animal models of human disease. These studies support RNAi as a new therapeutic approach with potential to change disease treatment. Clinical trials have recently begun, with RNAi therapeutic candidates under study for age-related macular degeneration (AMD) and respiratory syncytial virus (RSV) infection. RNAi involves the cleavage of target mRNA by the enzyme Dicer, leading to the formation of siRNA duplexes that are incorporated into the RNA-induced silencing complex (RISC). The guide strand of the siRNA is cleaved by Ago2, leading to mRNA down-modulation. Chemical modifications, such as phosphorothioate backbones and 2'-O-methyl modifications, enhance stability and potency of siRNAs. These modifications reduce immunostimulatory effects and improve nuclease resistance. Design considerations for potency and specificity include avoiding off-target silencing and immune stimulation. Bioinformatics methods, chemical modifications, and empirical testing are required to address these issues. The efficiency of the guide strand in incorporating into the RISC complex is a key factor in siRNA potency. The seed region of the siRNA is critical for specificity, and modifications in this region can reduce off-target effects. Local RNAi has shown efficacy in animal models for viral infections, ocular disease, nervous system disorders, cancer, and inflammatory bowel disease. Systemic RNAi has also demonstrated efficacy in various disease models, including hypercholesterolemia, rheumatoid arthritis, and viral infections. The use of chemical modifications and delivery strategies has improved the pharmacokinetic properties and cellular uptake of siRNAsRNA interference (RNAi) is a promising new class of pharmaceutical drugs that can specifically and potently inhibit disease targets. Traditional pharmaceutical approaches face challenges in identifying highly specific and potent drug candidates, especially for intractable targets. RNAi offers a powerful method for rapidly identifying inhibitors of disease targets across all molecular classes. Numerous proof-of-concept studies in animal models demonstrate the broad potential of RNAi therapeutics. The major challenge in drug development is identifying effective delivery strategies that can be translated to the clinic. Advances in this area and the initiation of clinical trials with RNAi therapeutic candidates suggest a potential transformation in modern medicine. RNAi is a fundamental cellular mechanism for silencing gene expression that can be harnessed for drug development. It can be used to reduce the expression of pathological proteins, including those difficult to modulate with traditional pharmaceutical approaches. RNAi therapeutics have the potential to significantly impact modern medicine. The mechanism involves the enzymatic cleavage of target mRNA, leading to decreased protein abundance. Synthetic siRNAs leverage the naturally occurring RNAi process, while viral delivery of shRNAs is an alternative strategy. This review provides an overview of the molecular mechanism of RNAi, the in silico design of siRNAs and shRNAs, chemical modifications that enhance stability, and strategies for cellular delivery. It summarizes the numerous publications demonstrating the robust efficacy of RNAi in animal models of human disease. These studies support RNAi as a new therapeutic approach with potential to change disease treatment. Clinical trials have recently begun, with RNAi therapeutic candidates under study for age-related macular degeneration (AMD) and respiratory syncytial virus (RSV) infection. RNAi involves the cleavage of target mRNA by the enzyme Dicer, leading to the formation of siRNA duplexes that are incorporated into the RNA-induced silencing complex (RISC). The guide strand of the siRNA is cleaved by Ago2, leading to mRNA down-modulation. Chemical modifications, such as phosphorothioate backbones and 2'-O-methyl modifications, enhance stability and potency of siRNAs. These modifications reduce immunostimulatory effects and improve nuclease resistance. Design considerations for potency and specificity include avoiding off-target silencing and immune stimulation. Bioinformatics methods, chemical modifications, and empirical testing are required to address these issues. The efficiency of the guide strand in incorporating into the RISC complex is a key factor in siRNA potency. The seed region of the siRNA is critical for specificity, and modifications in this region can reduce off-target effects. Local RNAi has shown efficacy in animal models for viral infections, ocular disease, nervous system disorders, cancer, and inflammatory bowel disease. Systemic RNAi has also demonstrated efficacy in various disease models, including hypercholesterolemia, rheumatoid arthritis, and viral infections. The use of chemical modifications and delivery strategies has improved the pharmacokinetic properties and cellular uptake of siRNAs