An integrated LC-MS and NMR metabolomic study was conducted to investigate metabolites whose formation was induced by lactofen (1), a soybean (Glycine max L.) disease resistance-inducing herbicide. First, LC-MS analyses of control and lactofen (1)-induced soybean extracts were performed. The LC-MS raw data were then processed by a custom designed bioinformatics program to detect the induced metabolites so formed. Finally, structures of unknown induced metabolites were determined on the basis of their 1D and 2D NMR spectroscopic data. Structure of two previously unreported compounds, 7,8-dihydroxy-4'-methoxy-3'-prenylisoflavone (2) and 7-hydroxy-4',8-dimethoxy-3'-prenylisoflavone (3) were elucidated together with four known prenylated compounds, 3'-prenyldaidzein (4), 8-prenyldaidzein (5), 3'-prenylgenistein (6), and 4-prenylcoumestrol (7). Compounds (2-6) are reported for the first time in soybean, as are the (13)C chemical shift assignments for compound (7). Formation of these six prenylated compounds was also induced by the primary defense glucan elicitor from the cell wall of the pathogen Phytophthora sojae (Kauf. and Gerde.), further suggesting a potential role in soybean defense. These results highlight the metabolic flexibility within soybean secondary product pathways and suggest that prenylation may be associated with defense responses. Moreover, this study demonstrates a promising future approach using metabolomics on elicitor-induced plants for discovery of unknown compounds even in relatively well studied plants.