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The K free light chain (K) index has been suggested as a reliable marker of intrathecal synthesis, despite the 2017 McDonald criteria for multiple sclerosis (MS) suggesting to “interpret with caution positive immunoglobulin G (IgG) index when testing for oligoclonal bands (OB) is negative or not performed”. The aim of this study was to compare the performance of K and IgG indexes for MS diagnosis and OB detection in a cohort of Italian patients. We enrolled 385 patients (127 MS, 258 non-MS) who had cerebrospinal fluid (CSF) analysis, including isoelectric focusing (IEF), to detect OB in the diagnostic work-up. Albumin, IgG and free light chains were measured by nephelometry and used to calculate IgG and K indexes. Although the two markers were highly related (r = 0.75, r² = 0.55, p < 0.0001), the K index showed greater sensitivity and negative predictive value (versus the IgG index) for OB detection (97% versus 48% and 97% versus 71%) and MS diagnosis (96% versus 50% and 98% versus 78%). These results support K index (and not IgG index) as a first-line marker for MS, followed by IEF, according to a sequential testing approach in CSF analysis.