SCOPE: Metabolites derived from individual foods found in human biofluids after consumption could provide objective measures of dietary intake. For comprehensive dietary assessment, quantification methods would need to manage the structurally diverse mixture of target metabolites present at a wide concentration range. METHODS & RESULTS: We developed a strategy for selection of candidate dietary exposure biomarkers, providing comprehensive coverage. An analytical method for 62 food biomarkers was validated by extensive analysis of chromatographic and ionization behaviour characteristics using triple quadrupole mass spectrometry. We used urine samples from two food intervention studies: one controlled, inpatient study (n = 19) and the other a free-living study where individuals (n = 15) were provided with food as a series of menu plans. As proof-of-principle, we demonstrated that the biomarker panel could discriminate between menu plans by detecting distinctive changes in the concentration in urine of targeted metabolites. We showed quantitative relationships between four biomarker concentrations in urine and dietary intake. CONCLUSIONS: We have demonstrated design concepts for an analytical strategy allowing simultaneous quantification of a comprehensive panel of chemically-diverse biomarkers of a wide range of commonly-consumed foods. We propose that integration of self-reported dietary recording tools with biomarker approaches will provide more robust assessment of dietary exposure. This article is protected by copyright. All rights reserved.