Purpose To characterize and analytically validate the MSDA Test, a multi‐protein, serum‐based biomarker assay developed using Olink® PEA methodology. Experimental design Two lots of the MSDA Test panel were manufactured and subjected to a comprehensive analytical characterization and validation protocol to detect biomarkers present in the serum of patients with multiple sclerosis (MS). Biomarker concentrations were incorporated into a final algorithm used for calculating four Disease Pathway scores (Immunomodulation, Neuroinflammation, Myelin Biology, and Neuroaxonal Integrity) and an overall Disease Activity score. Results Analytical characterization demonstrated that the multi‐protein panel satisfied the criteria necessary for a fit‐for‐purpose validation considering the assay's intended clinical use. This panel met acceptability criteria for 18 biomarkers included in the final algorithm out of 21 biomarkers evaluated. VCAN was omitted based on factors outside of analytical validation; COL4A1 and GH were excluded based on imprecision and diurnal variability, respectively. Performance of the four Disease Pathway and overall Disease Activity scores met the established acceptability criteria. Conclusions and clinical relevance Analytical validation of this multi‐protein, serum‐based assay is the first step in establishing its potential utility as a quantitative, minimally invasive, and scalable biomarker panel to enhance the standard of care for patients with MS.