The z-score method and its variants for testing mean difference are commonly used for hit selection in high-throughput screening (HTS) assays. Strictly standardized mean difference (SSMD) offers a way to measure and classify the short interfering RNA (siRNA) effects. In this article, based on SSMD, the authors propose a new testing method for hit selection in RNA interference (RNAi) HTS assays. This SSMD-based method allows the differentiation between siRNAs with large and small effects on the assay output and maintains flexible and balanced control of both the false-negative rate, in which the siRNAs with strong effects are not selected as hits, and the restricted false-positive rate, in which the siRNAs with weak or no effects are selected as hits. This method directly addresses the size of siRNA effects represented by the strength of difference between an siRNA and a negative reference, whereas the classic z-score method and t-test of testing no mean difference address whether the mean of an siRNA is exactly the same as the mean of a negative reference. This method can readily control the false-negative rate, whereas it is nontrivial for the classic z-score method and t-test to control the false-negative rate. Therefore, theoretically, the SSMD-based method offers better control of the sizes of siRNA effects and the associated false-positive and false-negative rates than the commonly used z-score method and t-test for hit selection in HTS assays. The SSMD-based method should generally be applicable to any assay in which the end point is a difference in signal compared to a reference sample, including those for RNAi, receptor, enzyme, and cellular function. © 2007 Society for Biomolecular Sciences.