Profiling nutritional biomarkers through human sample analysis is a powerful approach for understanding the intricate relationships between diet, nutrition, and health. This chapter provides a comprehensive overview of methodologies used to measure key nutritional biomarkers, including intake markers like Na+, K+, carotenoids, iron and lipid profiles, oxidative stress markers such as malondialdehyde (MDA) and F2-isoprostanes, genetic variants, and gut microbiota indicators. Detailed protocols are outlined for the accurate collection, preparation, and analysis of human biological samples, such as blood, urine, and feces, using advanced techniques like mass spectrometry, chromatography, HPLC, ELISA, and molecular assays.