A new method for extracting isoflavones from espresso coffee (EC) was coupled with high-performance liquid chromatography– tandemmass spectrometry (MS/MS) for the first time to analyse five isoflavones, which included both a glycosilated form, genistin and the aglycons daidzein, genistein, formononetin and biochanin A. Isoflavones were extracted fromcoffee samples usingmethanol, stored in a freezer overnight to precipitate proteic or lipidic residues and purified on SPE C18 cartridges before highperformance liquid chromatography–MS/MS analysis. The recovery percentages obtained by spiking the matrix at concentrations of 10 and 100 μg l1 with a standard mixture of isoflavones were in the range of 70 to 104%. The limits of detection and limits of quantification were in the range of 0.015–0.3 μg l1 and 0.05–1 μg l1, respectively. Once validated, the method was used to analyze the concentrations of isoflavones in six ECs and ten ground coffee samples. Only formononetin and biochanin A were found, and their respective concentrations ranged from 0.36 to 0.41 μg l1 and from 0.58 to 3.26 μg l1 in ECs and from 0.36 to 4.27 μgkg1 and from 0.71 to 3.95 μgkg1 in ground coffees. This method confirms the high specificity and selectivity of MS/MS systems for detecting bioactives in complex matrices such as coffee.