Introduction: Hepatitis B virus (HBV) infection is a significant global health concern with potential for silent transmission and severe outcomes. Timely and accurate detection of HBV surface antigen (HBsAg) is crucial for effective management and prevention of transmission. Methods: A cross-sectional study was conducted from January 2024 to March 2024 at a tertiary care center in central India. A total of 5439 blood samples were tested for HBsAg using both the rapid ICT method (Mediline-SA HBsAg Combo Rapid Test) and ELISA (Trustwell HBsAg ELISA Kit). Results: Among the samples tested, the rapid ICT method identified 112 HBsAg-positive cases and ELISA detected 105 positives. Using ELISA as the gold standard, the rapid ICT method showed a sensitivity of 99.04%, specificity of 99.85%, positive predictive value (PPV) of 92.85%, negative predictive value (NPV) of 99.98%, and diagnostic accuracy of 99.83%. Discussion: The study found ELISA to be more sensitive and specific compared to rapid ICT for HBsAg screening. Similar studies reported varying sensitivity and specificity rates for rapid ICT, highlighting the importance of method selection based on diagnostic requirements and resource availability. Conclusion: Despite its lower sensitivity, rapid ICT remains useful in resource-limited settings for initial screening due to its cost-effectiveness and ease of use. However, for definitive diagnosis and accurate detection of HBV, especially in asymptomatic cases and during blood screening, ELISA is recommended.