When studying gene function in vivo during development, gene expression has to be controlled in a precise temporal and spatial manner. Technologies based on RNA interference (RNAi) are well suited for such studies, as they allow for the efficient silencing of a gene of interest. In contrast to challenging and laborious approaches in mammalian systems, the use of RNAi in combination with oviparous animal models allows temporal control of gene silencing in a fast and precise manner. We have developed approaches using RNAi in the chicken embryo to analyze gene function during neural tube development. Here we describe the construction of plasmids that direct the expression of one or two artificial microRNAs (miRNAs) to knock down expression of endogenous protein/s of interest upon electroporation into the spinal cord. The miRNA cassette is directly linked to a fluorescent protein reporter, for the direct visualization of transfected cells. The transcripts are under the control of different promoters/enhancers which drive expression in genetically defined cell subpopulations in the neural tube. Mixing multiple RNAi vectors allows combinatorial knockdowns of two or more genes in different cell types of the spinal cord, thus permitting the analysis of complex cellular and molecular interactions in a fast and precise manner. The technique that we describe can easily be applied to other cell types in the neural tube, or even adapted to other organisms in developmental studies.