Vitamin A deficiency is a serious health problem in many developing countries. Provitamin A carotenoids in fruit and vegetables are the major source of vitamin A for a large proportion of the world's population. However, the contribution of plant foods is substantial only when both the consumption and provitamin A content of such food is high and, at the same time, the bioefficacy of the provitamin A is high. With respect to provitamin A carotenoids, the term bioefficacy is defined as the product of the fraction of the ingested amount that is absorbed (bioavailability) and the fraction of that which is converted to retinol in the body (bioconversion). Isotopic tracer techniques can meet the need for accurate and precise estimates of the bioavailability, bioconversion, and bioefficacy of dietary carotenoids in humans. Use of such techniques will enable proper evaluation of food-based approaches to eliminating vitamin A deficiency. In addition, the putative antioxidant capacities of carotenoids can be better understood if their bioavailability is known. Here, we discuss how tracer techniques can be applied to obtain reliable and representative data. A step-by-step discussion of aspects related to these techniques is provided, including study design, choice of isotopic tracers, dosing regimen, collection of samples, chemical analysis of samples, and data analysis.