2'-5'-Oligoadenylate synthetase plays a central role in the cellular innate antiviral response. Although activation of 2'-5'-oligoadenylate synthetase by double stranded RNA was discovered more than 30 years ago it is still unclear which sequence features are required by an RNA to activate the enzyme. A pool of chemically synthesized short double stranded RNAs of specific sequence was used to probe 2'-5'-oligoadenylate synthetase activation. It was found that activating double stranded RNAs contain the following motif: NNWWNNNNNNNNNWGN. Verification of this sequence motif in a pool of 102 small double stranded RNAs demonstrated a false positive prediction rate of 8% and a false negative prediction rate of 12%. The sequence motif identified provides mechanistic insight into the mechanism of 2'-5'-oligoadenylate synthetase activation by double stranded RNA and allows theoretical predictions whether a given RNA molecule has the capability to activate 2'-5'-oligoadenylate synthetase.