Abstract: BACKGROUND: North-West India has a high prevalence of the Thalassemia, and the Beta Thalassemia Trait often remains undetected in the population. Recognizing the need to address this public health issue, we initiated “Blood the Lifeline” project in 2006 with the aim to eradicate Thalassemia through proactive measures, including screening of voluntary blood donors. AIM: The primary aim of this study was to screen voluntary blood donors in an endemic area in northwestern India for Thalassemia trait. The study aimed to determine its prevalence, identify gender and ethnic group-wise distributions, and assess the implications for future interventions. METHODS: The screening program was conducted from January 2006 till December 2022. Voluntary blood donors were recruited from the target area and underwent Thalassemia trait screening. The screening process involved High Performance Liquid Chromatography using VARIANT II fully automated HPLC system to identify Thalassemia trait. Data regarding the total number of cases screened, gender distribution, ethnic group-wise distribution, and positive and negative cases were collected and analyzed. Screening Process: EDTA Pilot tube from each donor was collected and analyzed within 24 hrs of collection. RBC Counts, Hb Concentration, MCV, MCH, RDW-SD were noted to differentiate between Iron Deficiency Anaemia & thalassemia trait & following Criteria’s were taken into account: Total RBC Count: > 5.0 x 106 / micro-liter, Hb: < 11.7 g/dl, MCV: < 77 fl, MCH: < 28 pg & RDW-SD: < 45 fl. A combination of Formulas like Mentzer’s index= MCV/RBC <13; Shine and Lal Index (S&L) = MCV × (MCH/100) < 1530; Green & King (MCV2) × RDW]/ (Hb × 100) < 65 were used for screening. All the above formulas were introduced into the Blood Bank Software to exclude out the blood units requiring screening & confirmation. If any of the samples were positive on initial screening, then confirmatory test was done by demonstrating the elevation of concentration of HbA2 by HPLC VARIANT II fully automated analyser. RESULTS: A total of 377,503 voluntary blood donors for a 16 year period were screened and data was analysed .The Mean Hemoglobin of positive donors was 12.1 gm/dl; Mean RBC Count was 6.24 m/cu mm, MCV 67.79 fl, MCH 21.04 pg, RDW-SD 42.11fl & HbA2 was 7.48%. The majority of positive cases were found in the Sindhi (16.68%), Punjabi (10.32%), and Muslim (9.81%) ethnic groups. The Rajasthani population accounted for 6.01% of positive cases. CONCLUSION: This comprehensive screening program revealed a relatively high prevalence of Thalassemia trait in 2.29% of the screened population. The communities with higher prevalence (Sindhi, Punjabi, and Muslim) were educated about the disease and a targeted screening program will be further carried out in these ethnic groups. The data helped in planning genetic counseling, carrier screening, and pre-marital screening programs to reduce the transmission of Thalassemia in these populations. By focusing efforts on specific groups and locations with higher prevalence, a community based approach to prevent the birth of children with Thalassemia major is being implemented. It is also being used for analyzing the implications of different local policies pertaining to Thalassemia trait in the region. Collaborative efforts between healthcare organizations, community leaders, and governmental agencies are crucial to address the burden of Thalassemia and work towards its eradication. The results of this study serve as a foundation for future initiatives aimed at minimizing the impact of Thalassemia at a community level.