This article provides a comprehensive guide to using small interfering RNAs (siRNAs) for gene knockdown in mammalian somatic cells. RNA interference (RNAi) is a highly conserved mechanism that uses double-stranded RNA (dsRNA) to trigger the degradation of homologous mRNA, leading to specific gene silencing. The authors describe the protocols for designing and producing siRNA duplexes, which are 21- to 23-nucleotide duplexes that are highly effective in triggering sequence-specific mRNA degradation. They detail the selection of siRNA sequences, annealing procedures, and methods for assessing RNAi activity in mammalian cells. The article also includes protocols for transfecting siRNA duplexes into cells, monitoring gene knockdown, and validating the specificity of knockdown using immunofluorescence and Western blotting. The authors highlight the versatility of siRNAs in studying gene function, from assessing essential genes to analyzing specific cellular processes, and discuss their potential for therapeutic applications.This article provides a comprehensive guide to using small interfering RNAs (siRNAs) for gene knockdown in mammalian somatic cells. RNA interference (RNAi) is a highly conserved mechanism that uses double-stranded RNA (dsRNA) to trigger the degradation of homologous mRNA, leading to specific gene silencing. The authors describe the protocols for designing and producing siRNA duplexes, which are 21- to 23-nucleotide duplexes that are highly effective in triggering sequence-specific mRNA degradation. They detail the selection of siRNA sequences, annealing procedures, and methods for assessing RNAi activity in mammalian cells. The article also includes protocols for transfecting siRNA duplexes into cells, monitoring gene knockdown, and validating the specificity of knockdown using immunofluorescence and Western blotting. The authors highlight the versatility of siRNAs in studying gene function, from assessing essential genes to analyzing specific cellular processes, and discuss their potential for therapeutic applications.