With the increasing number of dystrophin replacement therapy trials, the need for reliable and accurate dystrophin quantification in muscle biopsies is becoming an important outcome measure. Quantification of dystrophin protein by mass spectrometry provides advantages over methods such as western blotting by offering improved precision, reproducibility, specificity, and a large quantifiable dynamic range. This protocol details a targeted mass spectrometry method for quantifying dystrophin in total muscle protein extracts using stable isotope-labeled dystrophin as a spike-in standard.