Mineral nutrient deficiencies are a worldwide problem that is directly correlated with poverty and food insecurity. The most common of these is iron deficiency; more than one-third of the world's population suffer from iron deficiency-induced anaemia, 80% of which are in developing countries. The consequences of iron deficiency include increased mortality and morbidity rates, diminished cognitive abilities in children and reduced labour productivity, which in turn stagnates national development. The developed world has made tremendous success in alleviating nutrient deficiencies through dietary diversification, food product fortification, improved public health care and supplementation. In developing countries, these strategies are often expensive and difficult to sustain, especially in rural areas. The rural poor typically consume what they grow and are dependent upon a small number of staple crops for the vast majority of their nutrition. Therefore, genetic improvement of staple crops (biofortification) is the most cost-effective and sustainable solution to this global health problem. In this study, we describe a strategy to enhance iron nutritional quality in maize using a human cell culture (Caco-2)-based bioassay as a phenotyping tool to guide genetic analysis of the trait. We also report validation of this approach using an animal feeding study.