Iron deficiency diagnosis is a key health priority during pregnancy. The precise determination of indicators is needed for the evaluation of iron deficiency. In the present study, we investigated the diagnostic features of hepcidin concentration as an iron deficiency assay during the second trimester of pregnancy. We collected 401 venous blood samples of pregnant females from 4 separate birthing hospitals. All the females were within 13-26 weeks of their pregnancy and without any comorbid conditions. The complete blood count, total iron binding capacity, ferritin, serum iron and serum hepcidin were determined. The women were categorized as being non-iron deficient (N-ID), iron deficient (ID), or with iron deficiency anemia (IDA). The mean hepcidin values for examined groups were, i.e., non-iron deficiency was 31.45±4.70 (μg/L), iron deficiency 20.47±2.48 (μg/L) and iron deficiency anemia was 17.33±1.90 (μg/L). The N-ID's hemoglobin mean levels were 13.05±0.10g/dL, ID 12.66±0.05g/dL and the IDA 8.11±00.12g/dL. In this article variations in hepcidin levels between N-ID, ID and IDA women are uncovered and it is reported that the lower hepcidin levels diagnosed in IDA are closely linked to hemoglobin in Pakistani women. Hence it is concluded that hepcidin can be a valuable marker in identifying iron deficiency and iron deficiency anemia during the second trimester of pregnancy, according to the Pearson's correlation data.