Exogenous application of double-stranded RNA (dsRNA) as RNA interference(RNAi)-based biopesticides represents a sustainable alternative to traditional transgenic, breeding or chemical-based crop protection strategies. While all eukaryotic organisms, a priori contain a functional RNAi pathway not all are effective targets of exogenously applied dsRNA. Key to understanding if an organism is a good target of this technology is understanding if functional small interfering RNAs (siRNAs) are produced from applied dsRNA. Traditional sequencing is ineffective as it is hampered by large amounts of degradation masking functional siRNA detection. Combining a novel biochemical purification technique and small RNA sequencing we have for the first time diagnosed whether functional siRNA species derived from exogenously applied dsRNA are present in many species including fungi, insects and nematodes. Further, we show in specific fungal species that siRNAs can be amplified and/or maintained over time, both facets of the pathway which contribute to effective and sustained RNAi. Our mechanistic dissection of the uptake and processing of exogenous dsRNA provides crucial insights into RNAi biopesticides and stands to add significant benefit to this emerging field of plant protection.