Having host range over 500 crop species, the soil borne necrotrophic fungus Macrophomina phaseolina, is one of the catastrophic factors in current agricultural sector. It infects plant during stressful environmental conditions such as hot and dry weather, resulting in alteration of plant growth, yield, and seed quality. In order to establish proper disease control system and develop disease resistant cultivars, we have established a pathosystem in the model plant, Medicago truncatula to study the molecular interactions between the host and the pathogen. In this study, we proposed to establish an unbiased quantitative assay using quantitative PCR (qPCR), which is more objective than other previously used approaches such as, scoring system based on percentage of necrosis and chlorosis seen on aerial part of plants or colony count approach. qPCR can quantitate the amount of fungal and plant DNA during infection over time, which are then used to understand disease progression by comparing amount of DNA from two different sources. This assay also facilitates disease diagnosis/assessment in the field.