This article contains the detailed biophysical characterization, biological testing, and photo-switching protocols of azobenzene containing siRNAs (siRNAzos), which have photoswitchable properties that can be controlled with light. First, the siRNAzos are characterized by annealing the sense and anti-sense strands together and then measuring the circular dichroism (CD) profile, and the melting temperatures (Tm ) of the duplexes. Second, the biological testing of the siRNAzos in cell culture is done to determine their gene silencing efficacy. Finally, their gene-silencing activities are measured after exposure to ultraviolet (UV) light in order to inactivate the siRNAzo, and then broadband visible light, which re-activates the siRNAzo. This inactivation/reactivation protocol can be done in real time, and is reversible and robust and can be performed multiple times on the same sample if desired. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Bio-physical characterization of siRNAzo duplexes Basic Protocol 2: Evaluation of azobenzene gene-silencing using Firefly Luciferase Basic Protocol 3: Evaluation of azobenzene gene-silencing using reverse transcriptase-polymerase chain reaction (RT-PCR).