Polymerized micelles obtained by photopolymerization of diacetylenic surfactants and which are forming polydiacetylenic systems (PDAs) have recently gained interest as stabilized monodisperse systems showing potential for the delivery of hydrophobic drugs as well as of larger biomolecules such as nucleic acids. Introduction of pH-sensitive histidine groups at the surface of the micellar PDA systems allows for efficient delivery of siRNA resulting in specific gene silencing through RNA interference. Here, we describe the detailed experimental procedure for the reproducible preparation of these photopolymerized PDA micelles. We provide physicochemical characterization of these nanomaterials by dynamic light scattering, transmission electron microscopy, and diffusion ordered spectroscopy. Moreover, we describe standardized biological tests to evaluate the silencing efficiency by the use of a cell line constitutively expressing the luciferase reporter gene