A multiplex real-time quantitative polymerase chain reaction (PCR) assay was developed to simultaneously detect the DNA of three rice planthoppers, that is, Sogatella furcifera (Horváth) (white-backed planthopper), Nilaparvata lugens (Stål) (brown planthopper) and Laodelphax striatellus (Fallén) (small brown planthopper), in the gut of their predators. The sets of primers and ALLGlo probes were targeted to the regions of internal transcribed spacer 2 (ITS2) genes in nuclear ribosomal DNA (rDNA). The sensitivity, specificity and interference test for the multiplex real-time quantitative PCR assay were analysed. The assay's detection limits were 100, 1000 and 100 copies for the white-backed planthopper, the brown planthopper and the small brown planthopper, respectively. The specificity tests showed no cross-reactivity with genomic DNA from 30 other dominant herbivores, saprophagous insects and predators from rice ecosystem for each planthopper species. The assay was used in a preliminary study of predation events on the three planthoppers by three major spiders viz., Pardosa pseudoannulata (Bösenberg et Strand), Ummeliata insecticeps (Bösenberg et Strand) and Tetragnatha maxillosa Thorell which each differ in their preferred microhabitat as well as their predatory habits in rice field, and the results showed their predation on each planthopper species could be well evaluated using this method. Therefore, the multiplex real-time quantitative PCR assay provides a new tool to study the mechanisms of prey shifting and natural regulation of the three rice planthoppers by generalist predators in rice ecosystem.