Much of the regulation of gene expression occurs at the level of protein synthesis. In addition to the canonical translation factors, a multitude of proteins and microRNAs (miRNAs) act as regulatory trans-acting factors. Mechanistic analysis of translational control benefits from functional cell-free systems that can be depleted of the responsible regulatory factors. Although antisense oligonucleotides facilitate the functional sequestration of the regulatory RNAs, immunodepletion of protein factors is technically challenging. Here we describe a simple and robust alternative protocol for the preparation of factor-depleted in vitro translation system derived from HeLa cells. The procedure relies on RNA interference-mediated knockdown of the factor of interest prior to extract preparation, and it overcomes problems with the availability and specificity of antibodies, as well as with the co-depletion of proteins associated with the factor under study. The complete procedure can normally be conducted within 1 week and carried out in parallel for multiple (candidate) factors.