BACKGROUND: Due to the challenges associated with accurate monitoring of dietary intake in humans, nutritional metabolomics (including food intake biomarkers) analysis as a complementary tool to traditional dietary assessment methods has been explored. Food intake biomarker assessment using postprandial dried blood spot (DBS) collection can be a convenient and accurate means of monitoring dietary intake vs 24-hour urine collection. OBJECTIVE: The objective of this study was to use nutritional metabolomics analysis to differentiate a high-fat, high-protein meat (HFPM) diet from a high-carbohydrate vegan (HCV) diet in postprandial DBS and 24-hour urine. DESIGN: This was a randomized controlled crossover feeding trial. PARTICIPANTS/SETTING: Participants were healthy young adult volunteers (n = 8) in California. The study was completed in August 2019. INTERVENTION: The standardized isocaloric diet interventions included an HFPM and an HCV diet. Participants attended 2 intervention days, separated by a 2-week washout. MAIN OUTCOME MEASURES: During each intervention day, a finger-prick blood sample was collected in the fasting state, 3 hours post breakfast, and 3 hours post lunch. Participants also collected their urine for 24 hours. DBS and urine samples were analyzed by ultra-performance liquid chromatography mass spectrometry to identify potential food intake biomarkers. STATISTICAL ANALYSES PERFORMED: Principal component analysis for discriminatory analysis and univariate analysis using paired t tests were performed. RESULTS: Principal component analysis found no discrimination of baseline DBS samples. In both the postprandial DBS and 24-hour urine, post-HFPM consumption had higher (P < 0.05) levels of acylcarnitines, creatine, and cis-trans hydroxyproline, and the HCV diet was associated with elevated sorbitol (P < 0.05). The HFPM diet had higher concentrations of triacylglycerols with fewer than 54 total carbons in DBS, and 24-hour urine had higher nucleoside mono- and di-phosphates (P < 0.05). CONCLUSIONS: Nutritional metabolomics profiles of postprandial DBS and 24-hour urine collections were capable of differentiating the HFPM and HCV diets. The potential use of postprandial DBS-based metabolomic analysis deserves further investigation for dietary intake monitoring.