This article contains the detailed synthesis and characterization protocols of azobenzene containing siRNAs, which have photoswitchable properties effectively controlled with light. First, the azobenzene scaffolds are synthesized via reduction of nitrophenyl alcohols in the presence of zinc. Next, the hydroxyl group of azobenzene derivatives are protected with a dimethoxytrityl (DMT) group, followed by phosphitylation with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite. These phosphoramidite monomers are compatible with automated solid-phase oligonucleotide synthesis to generate azobenzene-containing oligonucleotides. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of 4,4'-bis(hydroxymethyl)-azobenzene phosphoramidite Basic Protocol 2: Synthesis of 4,4'-bis(hydroxyethyl)-azobenzene phosphoramidite Basic Protocol 3: Synthesis, purification and characterization of oligonucleotides containing azobenzene derivatives.