Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays are a highly accurate and precise method for measuring transcript expression levels. A major drawback of RT-qPCR is the extensive optimization and validation necessary to produce high-quality assays, as described in the guidelines "Minimum Information for Publication of Quantitative Real-Time PCR Experiments." This chapter describes use of designed and optimized RT-qPCR assays that accurately detect expression of eight genes predicted to be centrally involved in the RNA interference (RNAi) pathways of western corn rootworm (WCR), and appropriate accompanying parameters. Assay gene targets include drosha, dicer-1, dicer-2, pasha, loquacious, r2d2, argonaute 1, and argonaute 2, and detection has been validated at nine different points in the WCR life cycle. These assays can be used with this procedure to assess expression of any one of these core RNAi pathway genes in up to 96 samples per 384-well qPCR plate.