Liquid chromatography/mass spectrometry (LC/MS) with atmospheric pressure chemical ionization has been successfully applied to the simultaneous analysis of free brassinosteroids (polyhydroxylated steroids that serve as plant growth regulators) as their naphthaleneboronates. Of a range of analytical conditions that were tested, optimum results were obtained by the use of reversed-phase high-performance liquid chromatography using a mixture of acetonitrile and water, The positive-ion spectra of naphthaleneboronates of brassinosteroids showed pseudo-molecular ions, [M + H](+), and fragment ions corresponding to the losses of hydroxyl groups of the steroidal nucleus. The [M + H](+) ion was the most abundant for brassinolide and the [M + H - H2O] + ions were the most abundant for castasterone, teasterone and typhasterol. Full-scan mass spectra were readily obtained from 400 ng of free brassinosteroids, while a limit of detection of 2 ng (signal-to-noise ratio 3) was attained in the selected-ion monitoring mode, The application of LC/MS to the study of brassinosteroids in a plant material Is demonstrated by the analysis of seeds of Cannabis sativa L.