Laetiporus sulphureus, commonly known as ”chicken of the woods”, due to its chicken-like flavor, texture, and aroma, is a species of mushroom with high potential as an alternative to meat, given its reported protein content of 11% to 21%. This study was conducted to determine the optimal substrate composition for efficient mycelial production of L. sulphureus for use as an alternative to meat. Two types of sawdust, Populus deltoides and Quercus acutissima, and eight nutrient sources were applied in single and mixed compositions. Analysis of mycelial growth revealed that the media containing P. deltodies and Q. acutissima, only supplemented with beet pulp, had the highest mycelial extension, of 106.7 mm and 101.3 mm, respectively. In addition, protein content analysis revealed that the combinations with the highest protein content in mycelia were cultivated on P. deltodies sawdust supplemented with a mixture of beet pulp and corn (21.3%), and on Q. acutissima sawdust supplemented with cottonseed hull (22.0%). The optimal substrate compositions identified in this study will serve as fundamental data for the large-scale production of L. sulphureus mycelia as an alternative to meat.