Because off-target effects hamper interpretation and validation of RNAi screen data, we developed a bioinformatics method, genome-wide enrichment of seed sequence matches (GESS), to identify candidate off-targeted transcripts in primary screening data. GESS analysis revealed a prominent off-targeted transcript in several screens, including MAD2 (MAD2L1) in a screen for genes required for the spindle assembly checkpoint. GESS analysis results can enhance the validation rate in RNAi screens.