Creating biofortified cultivars with increased grain zinc (Zn) content in localized staple food crops is a viable way to help the most vulnerable members of society who suffer from Zn deficiency and its related health effects. This means that in order to produce and spread nutrient-dense cultivars, micronutrient analytical techniques with low-cost, repeatable, and high throughput methodologies are required. While colorimetry (also known as the staining technique) is a straightforward, inexpensive method used for initial rapid screening, inductively coupled plasma optical emission spectroscopy (ICP) and atomic absorption spectrophotometry (AAS) are routinely used analytical techniques with high reproducibility and accuracy. The non-destructive technique known as X-ray fluorescence (XRF) spectroscopy has numerous advantageous features, including high throughput, low cost, multi-elemental, minimal or no sample preparation, and application over a broad concentration range from 100% to a few parts per million. Energy dispersive- XRF has been widely used for grain Zn analysis in rice, maize, wheat, pearl millet and beans. Comparative analyses have demonstrated good reproducibility and a strong correlation between the XRF results and ICP-OES; in rice, pearl millet, and wheat, (r2 = ≥0.95; P < 0.01). Based on XRF results breeders can easily discord the low-Zn early-stage breeding materials and the use of glass standards across XRF machines in the different parts of the world produces reproducible data like ICP. Therefore, this high throughput, cost-effective method accelerated large-scale screening of breeding lines in biofortification of different staple food crops in public and private sector programs. HarvestPlus, in association with the breeding resources program of CGIAR, coordinates the global XRF high-throughput screening and capacity building across CGIAR centers.