The soybean aphid, Aphis glycines, is a pest of soybeans in Asia and in recent years has caused extensive damage to soybean crops in North America. Within these agroecosystems, generalist predators form an important component of the assemblage of natural enemies, potentially reducing pest populations and exerting significant levels of regulation. Due to the complex nature of these food webs, molecular gut-content analysis is a valuable technique for examining trophic linkages in the field. We describe the development of molecular detection systems for examining the feeding behaviour of Orius insidiosus and the role of alternative prey, Neohydratothrips variabilis and Tetranychus urticae, and an intraguild predator, Harmonia axyridis, in soybean aphid food webs. Highly specific primer pairs were designed to all target prey and were used, for the first time, to examine these food webs in the field. Significant levels of predation on A. glycines were recorded while no evidence was gathered to suggest that important species of alternative prey disrupt pest consumption by O. insidiosus. These results confirm O. insidiosus as a valuable natural enemy of A. glycines in North American soybean agroecosystems.