Background and Purpose: In accord with ICH S7B guidelines, an in vitro human ether-a-go-go-related gene (hERG) assay is conducted to assess risk for delayed ventricular repolarization. Function of hERG could be affected by direct and acute mechanisms, or by indirect and chronic mechanisms. Oligonucleotides have unique mechanisms and time courses of action. To help frame the strategy of hERG assay for siRNA, investigation was performed to assess the time-course for siRNA-mediated inhibition of hERG function and gene expression. Experimental Approach: Commercially available siRNAs of hERG were evaluated in a stable hERG expressed cell line by whole-cell voltage clamp using automated electrophysiology and polymerase chain reaction (PCR). Key Results: In the acute hERG study, no effects were observed by applying 100 nM siRNA for 20 min. The chronic effects of 100 nM siRNAs on hERG function were evaluated by transfection and recorded over 8-48 hrs, after transfection. At 8 hrs there was no significant effect, while 81% reduction was observed at 48 hrs. Measurement of hERG mRNA levels demonstrated a 79% and 93% decrease of hERG mRNA at 8 and 48 hrs., respectively, consistent with a preceding effect on gene expression compared to protein function. Conclusion and Implications: Results presented here indicate that an anti-hERG siRNA requires a long exposure time (48 hrs.) in the hERG assay to produce a maximal effect; short exposures (20 min-8 hrs.) had no effect. These findings imply that oligonucleotides require specific and appropriate protocols to characterize their potential off-target effects on hERG channel.