Skeletal muscle is composed of different myofiber types with specific transcriptomic, proteomic, and metabolic identities. As such, skeletal muscle represents a valuable model to study cell differentiation and gene expression regulation. However, many molecular analyses of skeletal muscle are performed on whole muscle, with the caveat that many different cell types that constitute this organ contribute to the experimental results. Here, we describe a method to efficiently isolate individual myofibers from two muscle types and provide a guide to perform quantitative gene expression analysis of representative myosin heavy chain genes.