CE method with CuSO₄ and 2-hydroxypropyl-β-cyclodextrin additives in sodium acetate background electrolyte pH 4.3 for the simultaneous determination of amino acids and lactic acid was adapted for the comparative study of metabolism in “healthy” and non-alcoholic fatty liver disease model cells HepG2. In vitro model of the disease was developed by exposure HepG2 cells with oleate and palmitate to simulate an excessive flow of fatty acids into hepatocytes. The model was proven to be consistent with the disease pathophysiology, since intracellular triglyceride and cytokine interleukin 8 levels were increased, while cells viability was decreased. In order to check whether the metabolism of amino acids changes in pathology, we proposed sample preparation of culture medium and characterized the CE method by evaluating linear dynamic range, repeatability and intermediate precision of peak areas and migration times, accuracy (recovery rate and trueness estimated by reference method), detection limits and quantitation limits. The method proved to be sensitive, reliable and highly accurate for the quantitation of amino acids and lactic acid. The concentrations of amino acids in the culture medium of healthy and the disease model cells were measured and altered levels of Arg, Ala, Glu, Gln and lactic acid have been found in comparison to health control.