MicroRNAs (miRNAs) ubiquitously exist in microorganisms, plants, and animals, and appear to modulate a wide range of critical biological processes. However, no definitive conclusion has been reached regarding the uptake of exogenous dietary small RNAs into mammalian circulation and organs and cross-kingdom regulation. One of the critical issues is our ability to assess and distinguish the origin of miRNAs. Although periodate oxidation has been used to differentiate mammalian and plant miRNAs, validation of treatment efficiency and the inclusion of proper controls for this method were lacking in previous studies. This study aimed to address: 1) the efficiency of periodate treatment in a plant or mammalian RNA matrix, and 2) the necessity of inclusion of internal controls. We designed and tested spike-in synthetic miRNAs in various plant and mammalian matrices and showed that they can be used as a control for the completion of periodate oxidation. We found that overloading the reaction system with high concentration of RNA resulted in incomplete oxidation of unmethylated miRNA. The abundant miRNAs from soy and corn were analyzed in the plasma, liver, and fecal samples of C57BL/6 mice fed a corn and soy-based chow diet using our improved methodology. The improvement resulted in the elimination of the false positive detection in the liver, and we did not detect plant miRNAs in the mouse plasma or liver samples. In summary, an improved methodology was developed for plant miRNA detection that appears to work well in different sample matrices.