Small interfering RNAs (siRNAs) hold significant promise as therapeutic agents for treating human diseases. However, their use is often limited by unintended off-target effects arising from both the sense and antisense strands. Since the 5′-phosphate group on either strand is necessary for recognition by Argonaute 2 (AGO2), the core protein in the RNA-induced silencing complex (RISC), blocking phosphorylation of the sense strand may prevent its incorporation into RISC, thereby reducing potential off-target effects. In this chapter, we describe the use of biotin, a naturally occurring compound, to block 5′-end phosphorylation on either the sense or antisense strand to prevent their selection by AGO2. This strategy should offer a practical option to enhance siRNA specificity and efficacy.