Detection of plant viruses using enzyme-linked immunosorbent assay (ELISA) has proved to be among the most efficient method with high specificity. There are recurring improvements being explored for ELISA to optimize its sensitivity and specificity all the more (Tabatabaei and Ahmed, Cancer cell biology: methods and protocols. Springer, New York, 2022). ELISA is a powerful tool in plant virology due to its ability to detect and quantify specific viral proteins or antigens in plant tissues (Varma and Singh, Applied plant virology. Academic, 2020). This technique is crucial for several reasons. It provides a reliable and sensitive method for diagnosing viral infections, enabling early detection and accurate identification of viruses. ELISA is also valuable for monitoring the presence and concentration of viruses in plant populations, which helps in managing disease outbreaks and implementing control measures (Rodoni, Virus Res 141:150–157, 2009). Additionally, it aids in screening plants for viral infections before they are used in breeding programs or commercial production, thus preventing the spread of viruses (Rubio et al, Front Plant Sci 11:1092, 2020). Overall, ELISA’s precision and efficiency makes it an essential tool for maintaining plant health and ensuring agricultural productivity. This protocol provides an overview of the general procedure that is followed conventionally in all kinds of laboratory as well as the different types of adaptations commonly used for the purpose of plant virus detection.