Accurate non-invasive assessment of disease severity in patients with NAFLD is an urgent clinical need. A new proof-of-concept study demonstrates that a panel of 10 serum metabolites has high diagnostic accuracy and is superior to FIB-4 and the NAFLD Fibrosis Score (NFS) in detecting advanced fibrosis. The cross-sectional analysis included a prospective derivation cohort of 156 patients with biopsy-proven NAFLD and two validation cohorts, one including 142 participants who were assessed by MRI elastography (MRIE) and one including 59 patients with biopsy-proven NAFLD. In the derivation cohort, 32 of 652 assessed metabolites were associated with advanced fibrosis. The combined area under the receiver operating characteristic curve (AUROC) for the detection of advanced fibrosis with a panel of the top 10 metabolites was higher than that of FIB-4 and NFS (P = 0.002 and P = 0.017, respectively). The AUROC c-statistics of the panel were 0.94 and 0.84 for the MRIE and liver biopsy validation cohorts, respectively.