Introduction with Objectives: - The most common causes of microcytic hypochromic anemia in India are β Thalassemia & Iron deficiency anemia (IDA). β- Thalassemia is one of the most common single gene disorder in India with an overall prevalence of 3-4 %. Effective screening of β Thalassemia trait (βTT), decrease its incidence. Hb electrophoresis, serum iron profile & RBC indices are used to differentiate βTT from IDA .HbA2, red cell indices are observed as effective screening tests in βTT. Our main objective is to study the hemoglobin electrophoresis & RBC indices in β-Thalassemia Trait & to differentiate from IDA. Methodology: - A retrospective study of 50 patients (OP & IP) admitted in our KMC & hospital, Manipal was done. Study period was 1year (Aug 2015-Sep 2016). HbA2 level is studied by capillary zone electrophoresis method. Hb & red cell indices (MCV, MCH, RDW) were calculated. From red cell indices formulas –Mentzer (MI-cut off-13), Srivastava (SI-3.3), Shine & Lal(S&L-595), England & Frazer (E&F-1.39) were derived & serum iron profiles (Fe 2+, TIBC, Ferritin) was done to distinguish βTT from IDA. Vitamin B12 & folate assay was done . Sensitivity, specificity & Youden index were also calculated . Results:-All 50 pts were diagnosed as β-Thalassemia Trait (HbA2≥3.5 .Hb≤8gms/dl, MCV<60fL, MCH5x106/mm3, RDW>16%).Mean age group is 50yrs, M>F.3/50 cases (6%) shows ↑ HbF along with ↑HbA2.2/50 (4%) has ↑HbS .1 case shows IDA features with βTT. 49/50 (98%) cases of βTT showed-≤ cut off values of all index (MI,SI, S&LI ,E&FI). Serum iron profile was normal in 24/50 (48%), ↑ serum ferritin in 5/50 (10%).↑ vitamin B12 & folate levels seen in 9/50(18%).1 case showed ↓iron profile & ↑index. The MI is the most sensitive (>90%) & specific (>83%). Conclusion:-βTT & IDA are the M.C. causes of microcytic anemia. Hemogram parameters and RBC indices have significant role in βTT. HbA2, MCV & MI are the most sensitive tool to detect βTT.