Validated biomarkers are required to improve diagnosis, monitor disease progression and facilitate development of novel therapies. Protein biomarkers, discovered in multiplexed studies, proved however, to be difficult to implement in clinical use, due to the lack of analytical and clinical validation. In this paper, a novel orthogonal strategy is used to validate already identified serum biomarkers for Duchenne Muscular Dystrophy. Five out of ten disease progression biomarkers, previously identified by immuno-based proteomics methods, were validated using a mass spectrometry-based method. The biomarkers were analyzed by parallel reaction monitoring mass spectrometry assay in 72 longitudinal serum samples from 33 DMD patients and 12 healthy individuals. The serum concentration of carbonic anhydrase III (CA3) was measured between 346.5 and 12.2 fmol/ml in ambulant and non-ambulant DMD patients in contrast to 2.2 fmol/ml in healthy indviduals. Biomarker quantification using the PRM-MS method and an in-house developed sandwich immunoassay had a Pearson’s correlation of 0.88, demonstrating the feasibility in validating biomarkers using this strategy. This results indicate that future, orthogonal validation of DMD biomarkers is possible and reliable, providing means to assess and translate biomarkers to patient bed side.