Current methods for measuring fructan and arabinoxylan concentrations in wheat flour are time-consuming, and each type of the polymers requires a separate method. Here, we report the development of a new method that allows simultaneous determination of arabinoxylan and fructan contents in wheat flour. The new method is based on a single hydrolysis procedure for both arabinoxylans and fructans and an optimized separation technique for all monomers released. Owing to the use of milder hydrolysis conditions that afforded higher recovery of pentoses, the level of arabinoxylans in wheat flour determined by this new method is slightly higher than that measured with the widely used hydrolysis protocol. On the basis of the finding that, for a given flour sample, the total fructose concentration after hydrolysis is highly correlated with its total fructan concentration, the fructan content of a wheat flour sample can thus be estimated directly by the total fructose content. By simplifying and combining the two separate methods used for arabonoxylan and fructan analysis, this new method enables the quantification of arabinoxylans and fructans in wheat flour using a single acid hydrolysis step and a single high-performance liquid chromatography run.