India continues to grapple with a significant burden of HIV infections. Despite notable progress in prevention and treatment efforts, multiple challenges, such as high-risk populations, inadequate testing facilities, and limited access to healthcare in remote areas, persist. Though the Government of India offers HIV-1 plasma viral load testing at various medical centers, aiding treatment decisions and monitoring antiretroviral therapy effectiveness, enhancing care for individuals living with HIV under the National AIDS Control Program (NACP), the nation’s large population and diverse demographics further complicate its outreach and response. Hence, strategic interventions and alternative methods of testing remain crucial to curbing HIV transmission and improving the quality of life for those affected. Dried blood spot (DBS) sampling has emerged as a convenient and cost-effective alternative for HIV-1 viral load testing, revolutionizing the landscape of diagnostic and monitoring strategies for HIV infection. Though the plasma-based viral load remains the gold standard for monitoring HIV-1, DBS-based HIV-1 viral load testing holds immense promise for improving access to care, particularly in resource-limited settings where traditional plasma-based methods may be logistically challenging. DBS entails the collection of a small volume of blood onto filter paper, followed by drying and storage. This approach offers numerous advantages, including simplified sample collection, transportation, and storage, reducing the need for cold-chain logistics. Recent studies have demonstrated the feasibility and accuracy of DBS-based HIV-1 viral load testing, revealing a strong correlation between DBS and plasma measurements. Its implementation can enhance the early detection of treatment failure, guide therapeutic decisions, and ultimately contribute to better clinical outcomes for HIV-infected individuals. Hence, this review explores the principles, advancements, feasibility, and implications of DBS-based HIV-1 viral load testing.