Technical Abstract: Bikaverin is a polyketide metabolite produced by multiple species of the fungus Fusarium that infects plant crops, including maize. A technique was developed for the analysis of bikaverin by high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The quantitative nature of the LC-MS/MS experiment was demonstrated over a range of concentrations in maize. Limits of detection for the method were shown, and susceptibility of the method to matrix effects from maize was also evaluated. The method was applied to evaluate the ability of the maize pathogen Fusarium verticillioides to produce bikaverin in developing maize ears grown in an agricultural field.