BACKGROUND: The present study is designed to evaluate the reliability and cost effectiveness of cellulose acetate Hb electrophoresis and high performance liquid chromatography (HPLC) in the determination of HbA2 levels. METHODS: The test population comprised 160 individuals divided into four groups: normal individuals, beta-thalassemia trait (BTT) patients, iron deficiency anemia (IDA) patients, and co-morbid patients (BTT with IDA). HbA2 levels determined using cellulose acetate Hb electrophoresis and HPLC were compared. RESULTS: HbA2 levels were found to be diagnostic for classical BTT using either method. In co-morbid cases, both techniques failed to diagnose all cases of BTT. The sensitivity, specificity, and Youden's index for detection of the co-morbid condition was 69% and 66% for HPLC and cellulose acetate Hb electrophoresis, respectively. CONCLUSION: This study revealed that semi-automated cellulose acetate Hb electrophoresis is more suitable for use in beta-thalassemia prevention programs in low-income countries like Pakistan. This technique is easily available, simple and cost effective.