In arbuscular mycorrhizal symbiosis, the belowground mycelium that develops into the soil, not only provides extensive pathways for nutrient fluxes, the occupation of different niches, and dispersal of propagules, but also has strong influences upon biogeochemical cycling. By providing a valuable overview of expression changes of most proteins, shotgun proteomics can help decipher key metabolic pathways involved in the functioning of fungal mycelia. In this protocol, we describe the combination of extra-radical mycelium growth systems with gel-based extraction of fungal peptides amenable for shotgun protein profiling, which allows gaining information about the extra-radical proteome