AbstractPurpose: Non‐alcoholic fatty liver disease (NAFLD) is an important cause of chronic liver injury that has gained concern in clinical hepatology. The principal aim of this study was to find differences in protein expression between patients with NAFLD and healthy controls.Experimental design: Changes in protein expression of liver samples from each of the three groups of subjects, controls, non‐alcoholic steatosis, and non‐alcoholic steatohepatitis (NASH), were analyzed by DIGE combined with MALDI TOF/TOF analysis, a proteomic approach that allows to compare hundreds of proteins simultaneously.Results: Forty‐three proteins exhibiting significant changes (ratio ≥1.5, p<0.05) were characterized, 22 comparing steatosis samples versus control samples and 21 comparing NASH versus control samples. Ten of these proteins were further analyzed by Western blot in tissue samples to confirm the observed changes of protein expression using DIGE. The proteins validated were further tested in serum samples of different cohorts of patients.Conclusions and clinical relevance: Following this approach we identified two candidate markers, carbamoyl phosphate synthase 1 and 78 kDa glucose‐regulated protein, differentially expressed between control and NASH. This proteomics approach demonstrates that DIGE combined with MALDI TOF/TOF and Western blot analysis of tissue and serum samples is a useful approach to identify candidate markers associated with NAFLD, resulting in proteins whose level of expression can be correlated to a disease state.