['Anti interferon-beta (IFN-β) neutralizing antibodies (NAb) affect efficacy of treatment of multiple sclerosis patients, but exactly when the detrimental effects of NAbs offset therapeutic efficacy is debated. Quantification of intracellular pathway-specific phosphorylation by phospho-specific flow cytometry (phosphoflow) is a promising tool for evaluation of these effects in primary immune cells from treated patients at the single-cell level.', 'Samples for phosphoflow and gene expression changes were collected before administration of IFN-β and at four, six, and eight hours thereafter. Patients were NAb negative (n\u200a=\u200a3) or were NAb positive with low/medium (n\u200a=\u200a1) or high (n\u200a=\u200a2) NAb titers. Levels of phosphorylation of six Stat transcription factors (pStat) in seven cell subtypes and expression levels of 71 pathway-specific genes in whole blood were measured. The data was subjected to principal component analysis (PCA), fifty-fifty MANOVA, ANOVA, and partial least square regression (PLSR).', 'PCA of pStat levels clustered patients according to NAb class independently of time. PCA of gene expression data clustered patients according to NAb class but was affected by time and treatment. In the fifty-fifty MANOVA, NAb class was significant for both pStat levels and gene expression data. The ANOVA identified pStat1 protein in several cell subtypes as significantly affected by NAb class. The best fitting model for NAb prediction based on PLSR included pStat1 in monocytes, T cells, or lymphocytes and pStat3 in monocytes (r\u200a=\u200a0.97). Gene expression data were slightly less predictive of NAb titers.'] Does deficient phosphorylation of Stat1 in leukocytes identify neutralizing antibodies in multiple sclerosis patients treated with interferon-beta?