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Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and is associated with the worldwide epidemics of obesity, diabetes and cardiovascular diseases. NAFLD ranges from benign fat accumulation in the liver (steatosis) to non-alcoholic steatohepatitis (NASH), and cirrhosis which can progress to hepatocellular carcinoma and liver failure. Mass spectrometry and magnetic resonance spectroscopy-coupled stable isotope-based flux studies provide new insights into the understanding of NAFLD pathogenesis and the disease progression. This review focuses mainly on the utilization of mass spectrometry-based methods for the understanding of metabolic abnormalities in the different stages of NAFLD. For example, stable isotope-based flux studies demonstrated multi-organ insulin resistance, dysregulated glucose, lipids and lipoprotein metabolism in patients with NAFLD. We also review recent developments in the stable isotope-based technologies for the study of mitochondrial dysfunction, oxidative stress and fibrogenesis in NAFLD. We highlight the limitations of current methodologies, discuss the emerging areas of research in this field, and future directions for the applications of stable isotopes to study NAFLD and its complications.