Background: Molecular components in blood, like proteins, are used as biomarkers to reveal or predict disease states, guide clinical interventions and aid development of therapies. While multiplexing proteomics methods promote discovery of such biomarkers, it is generally difficult to translate them to clinical use due to lack of substantial evidence regarding their reliability as quantifiable indicators of disease state or outcome. To overcome this challenge, a novel orthogonal strategy is developed and used to assess reliability of biomarkers and analytically corroborate already identified serum biomarkers for Duchenne muscular dystrophy (DMD). DMD is a monogenic incurable disease characterized by progressive muscle damage currently lacking reliable and specific disease monitoring tools. Methods: Two technological platforms are used to detect and quantify the biomarkers in 72 longitudinally collected serum samples from DMD patients at 3 to 5 timepoints. Quantification of the biomarkers is achieved by detection of the same biomarker fragment either through interaction with validated antibodies in immuno-assays or through quantification of peptides by Multiple Reaction Monitoring Mass Spectrometry assay (PRM-MS). Results: Out of ten previously identified biomarkers by immuno-based proteomics methods, five are confirmed using the mass spectrometry based method. Two biomarkers, carbonic anhydrase III and lactate dehydrogenase B, are quantified with two independent methods, sandwich immunoassays and PRM-MS, to a Pearson correlation of 0.92 and 0.946 respectively. The median concentration of CA3 and LDHB in DMD patients is elevated in comparison to healthy individuals to 35- and 3-fold, respectively. Levels of CA3 vary between 10.26 and 0.36 ng/ml in DMD patients whereas that of LDHB vary between 15.1 and 0.8 ng/ml. Conclusions: These results indicate that orthogonal assays can be used to assess analytical reliability of biomarker quantification assays, providing means to facilitate translation of biomarkers to clinical practice. This strategy also warrants development of the most relevant biomarkers, markers that can be reliably quantified with different proteomics methods.