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Reticulocyte hemoglobin (Ret-Hb) content and soluble transferrin receptor (sTfR) are described as promising biomarkers in the analysis of iron status. However, the value of Ret-Hb and sTfR in the early detection of iron depletion, as frequently observed in children in high-income countries, is unclear. We hypothesized that young children to iron depletion, using the WHO cutoff of ferritin < 12 μg/l, would have lower Ret-Hb and higher sTfR concentrations compared to children with a ferritin ≥ level 12 μg/l.In this cross-sectional study, we analyzed mean concentrations of Ret-Hb and sTfR in 351 healthy children aged 0.5–3 years in a high-income country. The Student's t-test was used to compare Ret-Hb and sTfR concentrations between groups.We showed that concentrations of Ret-Hb and sTfR are similar in children with and without iron depletion. A decrease in Ret-Hb concentration was present only when ferritin concentrations were <8 μg/l. sTfR concentrations were similar in children with ferritin concentrations <6 μg/l and ≥ 12 μg/l.Our results showed that the discriminative value of Ret-Hb and sTfR for the detection of iron depletion is limited. Our findings suggest that ferritin is the most useful biomarker in the screening of iron depletion in healthy children in high-income countries. However, ideally, reference ranges of iron status biomarkers should be based on studies showing that children with concentrations outside reference ranges have poor neurodevelopmental outcomes.