The application of genome scale RNA interference (RNAi) relies on the development of high quality RNAi high throughput screening (HTS) assays. An important quality control (QC) characteristic in an HTS assay is how well the positive controls, samples, and negative controls can be separated from each other in the assay. Signal-to-noise ratio, signal-to-background ratio, Z-factor and Z’-factor have been adopted to evaluate this QC characteristic in small molecule HTS assays. However, none of them have clear probability meanings and strong statistical bases. A pair of easily interpretable and theoretically based metrics, strictly standardized mean difference (SSMD) and coefficient of variability in difference (CV D), were proposed for measuring population separation. In this paper, we investigated the use of SSMD along with CV D to evaluate the quality of RNAi HTS assays and compared it with other measures. Applying this pair of metrics to an in-house RNAi HTS assay demonstrated that SSMD is superior to signal-to-noise ratio and Z’-factor for QC evaluation. The cutoff criterion of SSMD has a good theoretical basis. Therefore, SSMD has the capacity to become a generally accepted QC metric in HTS assays.