Neurofilament light chain (NfL) has emerged as a promising biomarker for detecting and monitoring axonal injury. Until recently, NfL could only be reliably measured in cerebrospinal fluid, but digital single molecule array (Simoa) technology has enabled its precise measurement in blood samples where it is typically 50 100 times less abundant. We report development and multi-center validation of a novel fully automated digital immunoassay for NfL in serum for informing axonal injury status. Methods: A 45-min immunoassay for serum NfL was developed for use on an automated digital analyzer based on Simoa technology. The analytical performance (sensitivity, precision, reproducibility, linearity, sample type) was characterized and then cross validated across 17 laboratories in 10 countries. Analytical performance for clinical NfLmeasurement was examined in individual patients with relapsing remitting multiple sclerosis (RRMS) after 3 months of disease modifying treatment (DMT) with fingolimod. Results: The assay exhibited a lower limit of detection (LLoD) of 0.05 ng/L, a lower limit of quantification (LLoQ) of 0.8 ng/L, and between-laboratory imprecision <10 % across 17 validation sites. All tested samples had measurable NfL concentrations well above the LLoQ. In matched pre post treatment samples, decreases in NfL were observed in 26/29 RRMS patients three months afterDMT start, with significant decreases detected in a majority of patients. Conclusions: The sensitivity characteristics and reproducible performance across laboratories combined with full automation make this assay suitable for clinical use for NfL assessment, monitoring in individual patients, and crosscomparisons of results across multiple sites.