Some mushroom proteins and peptides have reported biological activities increasing interest due to their potential as nutraceuticals. It is necessary to research optimizing the substrates for biomass and protein production to improve the biotechnological approaches. This study evaluated the effect on biomass production and total intra-protein in Lentinula edodes grown by liquid-state fermentation (LSF) for eight culture mediums. The higher total protein content was found on bienestarina media (BIE), glucose, peptone, and yeast extract media (GPY). The protein profile using a shotgun proteomics approach by LC-MSMS showed the media's marked influence on protein production. Most proteins identified using the BIE medium did not appear when the mushroom grew in the GPY. Some of the detected proteins included lectins and enzymes, are used in industrial applications. These results would indicate that BIE promotes in L. edodes the production of proteins with potential for its application to the biotechnology industry