Piriformospora indica is a mycorrhiza like fungus of the order Sebacinales,which colonizes root and forms symbiosis with almost all terrestrial plants on earth. Information about the P. indica quantitative proteomics is limited.This limits our understanding of its multitrophic interaction with plants. A quantitative proteomic analysis of the fungus vis a vis its interaction with rhizospheric bacteria and the plant can reflect on the underlying proteins/enzymes. We describe a protocol for efficient extraction of cellular proteins from the fungus for two-dimensional gel electrophoresis following interaction with Azotobacter chroococcum in axenic culture. Polysaccharides and abundant salts present in Hill and Kaefer broth hinders extraction of protein from fungal biomass. Good quantity and quality of proteins are required for two-dimensional gel electrophoresis and proteomic analysis. We could use the extracts for proteomic analysis following a high resolution of 2-DE reference map of the fungus after interaction with A. chroococcum.