Several clinical trials are working on drug development for Duchenne and Becker muscular dystrophy (DMD and BMD) treatment, and, since the expected increase in dystrophin is relatively subtle, high-sensitivity quantification methods are necessary. There is also a need to quantify dystrophin to reach a definitive diagnosis in individuals with mild BMD, and in female carriers. We developed a method for the quantification of dystrophin in DMD and BMD patients using spectral confocal microscopy. It offers the possibility to capture the whole emission spectrum for any antibody, ensuring the selection of the emission peak and allowing the detection of fluorescent emissions of very low intensities. Fluorescence was evaluated first on manually selected regions of interest (ROIs), proving the usefulness of the methodology. Later, ROI selection was automated to make it operator-independent. The proposed methodology correctly classified patients according to their diagnosis, detected even minimal traces of dystrophin, and the results obtained automatically were statistically comparable to the manual ones. Thus, spectral imaging could be implemented to measure dystrophin expression and it could pave the way for detailed analysis of how its expression relates to the clinical course. Studies could be further expanded to better understand the expression of dystrophin-associated protein complexes (DAPCs).