Plasma concentrations of amino acids (AAs), in particular, branched chain AAs (BCAAs), are often found increased in nonalcoholic fatty liver disease (NAFLD); however, if this is due to increased muscular protein catabolism, obesity, and/or increased insulin resistance (IR) or impaired tissue metabolism is unknown. Thus, we evaluated a) if subjects with NAFLD without obesity (NAFLD‐NO) compared to those with obesity (NAFLD‐Ob) display altered plasma AAs compared to controls (CTs); and b) if AA concentrations are associated with IR and liver histology. Glutamic acid, serine, and glycine concentrations are known to be altered in NAFLD. Because these AAs are involved in glutathione synthesis, we hypothesized they might be related to the severity of NAFLD. We therefore measured the AA profile of 44 subjects with NAFLD without diabetes and who had a liver biopsy (29 NAFLD‐NO and 15 NAFLD‐Ob) and 20 CTs without obesity, by gas chromatography–mass spectrometry, homeostasis model assessment of insulin resistance, hepatic IR (Hep‐IR; Hep‐IR = endogenous glucose production × insulin), and the new glutamate–serine–glycine (GSG) index (glutamate/[serine + glycine]) and tested for an association with liver histology. Most AAs were increased only in NAFLD‐Ob subjects. Only alanine, glutamate, isoleucine, and valine, but not leucine, were increased in NAFLD‐NO subjects compared to CTs. Glutamate, tyrosine, and the GSG‐index were correlated with Hep‐IR. The GSG‐index correlated with liver enzymes, in particular, gamma‐glutamyltransferase (R = 0.70), independent of body mass index. Ballooning and/or inflammation at liver biopsy were associated with increased plasma BCAAs and aromatic AAs and were mildly associated with the GSG‐index, while only the new GSG‐index was able to discriminate fibrosis F3‐4 from F0‐2 in this cohort. Conclusion: Increased plasma AA concentrations were observed mainly in subjects with obesity and NAFLD, likely as a consequence of increased IR and protein catabolism. The GSG‐index is a possible marker of severity of liver disease independent of body mass index. (Hepatology 2018;67:145‐158).