Aim: Iron deficiency anemia (IDA) is a common medical condition, yet there is still some diagnostic uncertainty in this respect. The aim of this study was to compare the clinical significance of biomarkers of iron deficiency (ID) in diagnosing IDA and iron-deficient erythropoiesis in anemic patients. Materials and methods: A total of 103 untreated patients with non-hemolytic anemia were included. Blood count, reticulocyte hemoglobin content (CHr), iron, transferrin saturation (TSAT), ferritin (Ferr), soluble transferrin receptor (sTfR) and sTfR/logFerr index (sTfR-F index) were determined in the patients. Results: TSAT<16% diagnosed 79 patients with IDA (76.6%), Ferr<30 µg/l - 50 patients with IDA (48.5%). Thomas-plot analysis found 76 patients with ID (73.8%) and 56 of them were with iron-restricted erythropoiesis and IDA (54.4%). Biomarkers of ID were significantly different in anemic patients with iron-deficient erythropoiesis (CHr<28 pg) compared with patients with normal hemoglobinisation (p<0.001). With regard to the predictive value of the parameters of ID for iron-deficient erythropoiesis in anemia, their mutually controlled influence proved sTfR-F index only as independent statistically significant (p=0.011). The optimal cut-off value of sTfR-F index from the ROC curve analysis for detecting iron-deficient erythropoiesis in anemia (CHr<28 pg) was 1.35, with sensitivity of 82.1% and specificity of 80.9% (AUC 0.866; p<0.001). Conclusions: Diagnosis of IDA depends on the applied biomarkers of ID, and TSAT or ferritin when used alone may lead to diagnostic difficulties. Combining sTfR-F index and CHr to evaluate iron-deficient erythropoiesis in patients with anemia in addition to ferritin and TSAT could contribute to improving the diagnosis of IDA in clinical practice.