Pomiferin and osajin are major bioactive isoflavones found in Maclura pomifera (Rafin.) Schneider (Moraceae). A simple, reproducible, and sensitive method for analysis of pomiferin and osajin in different parts of M. pomifera has been developed and validated. The compounds were separated on a C(8) column by gradient elution at a flow rate at 1.0 mL min(-1). Column eluent was monitored at 280 nm by use of a diode-array detector. Linear range, peak purity, selectivity, system performance, precision, accuracy, and robustness were shown to be acceptable. LC-MS with atmospheric pressure electrospray ionization was used for identification of the compounds; use of positive-ion mode resulted in protonated pseudomolecular ions of the compounds.