Hepatic steatosis, accumulation of intracellular triglyceride (TG) (>5% of hepatic tissue), is the prerequisite for the development of non-alcoholic fatty liver disease (NAFLD). NAFLD represents a spectrum of liver diseases and is associated with obesity and obesity-related metabolic diseases such as type 2 diabetes and cardiovascular disease. Steatosis occurs due to an imbalance between fatty acid input and removal (fatty acid partitioning) which can be affected by genetics, diet, hormones and drugs; making the cause of NAFLD difficult to elucidate. Available cell models provide insight however, development of human cell models that are characterized in terms of culture conditions and the effect this may have on intracellular fatty acid partitioning are required. We set out develop a model of TG accumulation using a physiological mixture of exogenous fatty acids (representative of dietary or non-esterified fatty acids) and assess fatty acid partitioning in a novel human liver cell line (LIVOAPOLY). Under basal conditions LIV0APOLY cells do not have notable lipid accumulation. Short-term exposure of exogenous fatty acids promoted storage, oxidation and secretion of TG. Additionally, when exposed to higher glucose concentrations intracellular TG content increased, indicative of an increase in de novo lipogenesis (DNL) as would be predicted in vivo. Stable isotopes were used to assess the fate of exogenous fatty acids in LIV0APOLY cells compared to primary human hepatocytes, the gold standard liver cell model. Both cell types stored similar amounts of labelled fatty acids TG, however, oxidation and secretion of FA varied between the cell types. The differences observed maybe due to variations in culturing formulations between the cells. LIV0APOLY cells can be considered a useful human liver cell model for studying the effects of exogenous metabolic substrates on fatty acid partitioning in order to delineate the mechanisms which may influence the development and progression of NAFLD.