This study presents a strategy based on repeatable reversed-phase LC-TOF-MS methods and chemometric statistical tools, including untargeted PCA and supervised OPLS-DA models, to identify low-yielding compounds with potent immunostimulant activity in Phaleria nisidai (Thymelaeaceae), a plant with a history of use as an adaptogen on the islands of Palau in Micronesia. IFNγ ELISA assays were used to classify chromatographic fractions according to imunomodulatory activity prior to LC-TOF-MS chemometric analysis to target and identify compounds likely to contribute to observed activity. Simplexin, a daphnane diterpene ester, was identified for the first time from this genus and caused an increase in the production of cytokines (IFNγ, IL1β, IL6, and IL13) by peripheral blood mononuclear cells. Five other daphnane diterpene esters were tentatively identified for the first time from this plant based on mass spectral data and are marker metabolites distinguishing active from inactive fractions. This analytical approach increased the efficiency of bioactivity-guided fractionation and has the potential to minimize redundant isolation and identify minor constituents with potent activity from a complex matrix.