A rapid, HPLC-based screening procedure for the main classes of secondary metabolites in Nicotiana attenuata leaves (alkaloids, phenolics, and diterpene glycosides) is reported. In a single step, leaves are extracted in aqueous acidified (0.5% acetic acid) methanol, and the extracted compounds are separated by reversed-phase HPLC with an acidic water/acetonitrile gradient in <30 min. The utility of the method in quantifying changes in the secondary metabolites after methyl jasmonate treatment of the plants, a treatment known to elicit resistance to herbivores in nature, is illustrated. Methyl jasmonate treatment elicited dramatic increases in some secondary metabolites (caffeoylputrescine, nicotine, and diterpene glycosides increased 12.5-, 1.4-, and 1.9-fold, respectively) but left others, such as rutin, unchanged. Such broad-based analytical screens will help characterize environmental and genetic changes in secondary metabolite profiles.