In Drosophila cells, RNA interference (RNAi) can be triggered by synthetic long double-stranded RNAs (dsRNAs). For many Drosophila cell lines and cell types, passive dsRNA uptake is inefficient. More complete silencing responses can often be obtained in Drosophila S2 cells using transfection, perhaps because higher levels of intracellular dsRNA are achieved. In this protocol, S2 cells are transfected with dsRNA using QIAGEN's Effectene reagent, which has proven to be reliable for many investigators. A plasmid DNA can also be included in the transfection mix to provide additional functionality. The plasmid DNA can encode, for example, a reporter of the activity of a pathway or specific transcription factor, or a marker that allows visualization of some cellular behavior or structure. It is also useful to include a plasmid that encodes a fluorescent protein simply to monitor transfection efficiency.