Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle loss and weakness caused by the lack of dystrophin. Dystrophin protein expression could be a biomarker to evaluate drug efficacy that recovers dystrophin levels in patients, including exon skipping and micro dystrophin. Currently, a semi-quantitative assay using western blots has been used. However, the current methods have sensitivity, quantifiability and reproducibility limitations. To address these issues, we established a highly sensitive and quantitative assay using the SMCxProTM and recombinant full-length dystrophin as a reference standard. Buffer-based lower limit of quantitation (LLOQ) of the assay is 1.2 nM and is 65-fold higher than that of MSD (75.5nM). The minimum required dilution factor is 8-fold, and the spike recovery rate is 107% determined by using patient muscle homogenate samples. In conclusion, dystrophin levels can be measured in individual muscle samples of non-DMD (n=9) and DMD participants (n=8). Dystrophin levels in non-DMD and DMD patient muscle are 93.2±31.9 fmol/total protein mg (range: 49.4-145.3) and 14.5±6.8 fmol/total protein mg (range: 6.18-22.6), respectively. Dystrophin levels in all samples are above of LLOQ, and the lowest concentration of dystrophin in DMD samples is two-times higher than LLOQ. These data suggest that our highly sensitive-quantitative assay is useful to accurately evaluate drug efficacy that is designed to increase dystrophin.