Reticulocytes are immature, non-nucleated RBCs that still contain RNA. It provides useful information about the capacity of the bone marrow to synthesize and release red cells in response to anemia and helps to distinguish between decreased RBC production and enhanced peripheral destruction. A prospective study was done between thalassemia and iron deficiency anemia cases. The duration of the study was 1 year where 139 cases were included. In our study, a total of 139 patients are included out of which 94 cases were of Thalassemia and 45 cases of Iron deficiency anemia. Measurement of complete blood count (CBC) with reticulocyte fractions was done in the SYSMEX XN-550 system and for serum ferritin, Architect iSystem was used. This study demonstrates the cut-off value of IRF for detecting Thalassemia trait as 15.85-21.50%, IDA was >21.50% and for Thalassemia major it was < 15.85%. The LFR% optimum cut-off for detecting Thalassemia trait was estimated to be 83.1-84.2%, IDA <83.1%and for Thalassemia major it was > 84.2%. The MFR% optimum cut-off for detecting Thalassemia trait was estimated to be <12.9%, IDA was >13.25% and for Thalassemia major it was 12.9-13.25%. The HFR% optimum cut-off for detecting Thalassemia trait was estimated to be 2.4-5.3%, IDA was >5.3%and for Thalassemia major it was < 2.4%. The use of new hematological parameters and doing further comparative-based studies will help in differentiating various types of anemia and give knowledge of physiopathology of the underlying disease.