RNA interference (RNAi) has become a widely used tool to analyze biological functions in vivo and in vitro. With the availability of an increasing number of Drosophila cell lines, a variety of different processes can be studied ranging from cell cycle control defects to signaling pathway activities and changes in cell morphology. Owing to the ease of RNAi in Drosophila cells, this experimental system has become a preferred method to screen for novel cellular factors, before their in depth analysis. We here describe the experimental procedures for RNAi experiments in cultured Drosophila cells, starting from the design of long double-stranded RNAs, their synthesis by in vitro transcription and application for cell-based RNAi experiments from low to high-throughput formats. Finally, we show how phenotype analysis can be performed using cell-based assays for luminescence or flow cytometric analysis as examples.