Macrophomina phaseolina (M. phaseolina) is a soil-borne necrotrophic fungus that causes charcoal rot disease. The pathogen has very wide host range (up to 500 plant species) and affects seed quality, plant growth and yield. No effective management approach is developed that can control the disease, and no resistant cultivars are available for most crop species. In our laboratory, we have developed a pathosystem using the model plant Medicago truncatula (M. truncatula). This helps in identifying the potential host genes to develop resistant cultivars by studying molecular interactions between the pathogen and its plant hosts. To characterize the disease symptoms and progression, a scoring system based on necrosis and chlorosis that appear on the above-ground part of the plant, was developed. Although it is an easy and quick approach to analyze disease progression in any plant, there exist many limitations in such technique. So, to overcome those limitations we had developed a quantitative method to directly measure the pathogen biomass during infection process using quantitative real-time PCR (qPCR), which has become a frequently used method in disease analysis due to its specificity, accuracy and sensitivity. This approach allowed us to measure the amount of fungal genomic DNA which correlates with fungal biomass in infected plant tissue and soil samples. With qPCR data obtained, two approaches were developed and analyzed using student t-test to find any statistical significance. Both approaches were correlated with the disease symptoms seen during root harvest. By comparison, the assay based on the ratios between fungal genomic DNA and host genomic DNA over the course of infection provides a more consistent and sensitive assay than other approaches.