Reliable information about the nutritional status is essential to identify potential critical nutrients and the population groups at risk of deficiency, as well as to develop effective public health policies to counteract unfavorable nutrition patterns that contribute to morbidity and mortality. In this review, the important role of biomarkers in the assessment of nutritional status is outlined, major strengths and limitations of established and new biomarkers are described, and important criteria for biomarker selection and development are discussed. Indeed, biomarkers offer a more objective assessment tool than pure dietary approaches that suffer from inadequate data reporting in particular, although biomarkers are often only measured in subsamples because of the higher costs and proband burden they entail. However, biomarkers are subject to individual variability and influences from other factors besides the nutrient of interest. Rapid turnover or tight control of nutrient concentrations in blood (homeostasis) limits their sensitivity as biomarkers, as in the case of many trace elements. The existence of different forms of a micronutrient in the body adds additional complexity. Functional biomarkers, such as enzyme activities, mirror long-term status better but are subject to confounding factors, and some are influenced by several micronutrients, not specific for only 1, so using a combination of biomarkers is advisable. Additionally, the applicability of a biomarker also depends on the existence of adequate reference values and cutoff points for the target population. Therefore, a careful selection is warranted, especially when biomarkers are to be used in larger samples.