SDS-PAGE Western blots allow for the identification of the presence and relative quantity of proteins of interest on the basis of their molecular weight and their recognition by specific antibodies. This technique has historically been useful in the diagnosis and study of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), as it allows the recognition of clinically relevant abnormalities in the amount or size of dystrophin. While this technique is used infrequently for diagnosis at this point (as genetic testing for pathogenic variants of the DMD gene has become standard practice), it remains an important tool in basic science research and in the evaluation of therapies that restore or replace dystrophin expression. This chapter provides a technique that is suitable for the quantitative assessment of dystrophin expression in isolates of skeletal muscle tissue, based on our experience and guidance provided by the US Food and Drug Administration (FDA) for these types of assays. Topics associated with the design of quantitative Western blot techniques, options for loading control use, and the challenges associated with variable pathology within specimens are also discussed. Performing this technique can provide reproducible and reliable quantification of full-length or shortened dystrophin protein content in frozen muscle tissue.