Introduction: With each donation of whole blood, healthy blood donor loses 200 – 250 mg of iron. This loss is compensated by the mobilization of stored iron. Several studies showed that blood donors may be at risk of iron deficiency/depletion and the development of anemia. This risk is particularly pronounced in female donors of childbearing age and frequent blood donors. Therefore, implementation of numerous measures are considered in order to prevent iron deficiency and improve the safety of blood donors. Aim: The aim of this study was to evaluate the iron status of voluntary blood donors in Croatia, primarily by measuring ferritin level in donors with different donation freqency, to assess the diagnostic value of RBC parameters, as well as combined cell index (CCI) in detecting reduced iron stores, to evaluate the blood donor safety in relation to the iron stores using current selection criteria and to consider the implementation of measures to prevent iron deficiency, particularly the introduction of ferritin testing in blood donors. Methods: The study involved 1,084 male and 792 female whole blood donors, accepted for blood donation. In addition to the short questionnaire about general data and lifestyle, two additional samples were collected: one for the determination of complete blood count (CBC) and one for the determination of ferritin, serum iron, UIBC/TIBC. Statistical analysis was performed using the MedCalc software and Excel 2016. Results: In male donors, linear drop of serum ferritin was demonstrated with increasing frequency of blood donations:from the median of 129 μg/L in the control group to 43 μg/L in those with 7 – 8 donations in two years. In control group of female donors, the median value of ferritin was 33 μg/L, while in test groups the median ferritin was within a narrow range between 22 μg/L and 29 μg/L, and in correlation with the menstrual status. Depleted iron stores (ferritin ˂12 μg/L) were found in males with the frequency of 0% in the control group and 1.95% in those with 1 – 8 donations in two years. Depleted iron stores were found in 6.7% females of the control group and in 17.2% of female donors with 1 – 6 donations in two years. Combined Cell Index (CCI) showed satisfactory diagnostic value in the assessment of depleted iron stores in both sexes. Conclusion: In addition to more intense education of blood donors on the importance of iron and its loss during the blood donation, we propose the implementation of quantitative method for haemoglobin determination, along with the gradual introduction of ferritin test.