Dystrophin quantification by western blot has been a mainstay of dystrophinopathy research for decades. More recently, the capillary western immunoassay has emerged as an alternative to traditional western blots. With this technique, electrophoresis and detection steps all take place in glass capillaries instead of gels and membranes. Advantages include the ability to perform quantification on very small amounts of protein lysate, as well as high-throughput and reproducibility. We provide here an optimized protocol for capillary western quantification of dystrophin expression, as well as validation data characterizing the performance of the assay.