Expansion of fibrous connective tissue and abnormal deposition of extracellular matrix (ECM) are at the basis of many fibrotic diseases. Fibrosis can occur in response to both physiological and pathological cues, including wound healing, tissue remodeling/repair and inflammation. Chronic fibrosis can lead to severe tissue damage, organ failure and death. Assessing the extent of organ fibrosis is crucial for accurate diagnosis of this condition. The use of Masson's trichrome staining of tissue sections from skeletal muscle is a fast method for detection of morphological alterations indicative of a fibrotic phenotype in this organ. This staining method detects the extent of collagen fibers deposition and, because it employs the combination of three dyes, can also distinguish muscle fibers (red), from collagen (blue) and nuclei (black), simultaneously.