Imaging of dystrophin and related proteins using light microscopy is a key assay in neuromuscular pathology diagnostic and research laboratories. Immunofluorescence co-staining of dystrophin alongside markers like laminin or its glycoprotein binding partners allows for quantitative analysis of these markers in relation to each other, enabling the reporting of metrics like the percentage of dystrophin-positive fibers and dystrophin staining intensity at the sarcolemma. When designed carefully, automation of image analysis approaches allows for higher throughput, greater precision, and less bias than manual counting of muscle fibers. These automated approaches are especially valuable for rigorously and objectively testing the efficacy of potential therapeutic strategies in development for restoring dystrophin expression. This chapter presents a validated method, its modifications, and some alternatives for automated quantitative analysis of immunofluorescence images of dystrophin or other muscle markers co-stained with a sarcolemmal marker for muscle fiber detection.