We present a liquid chromatography/mass spectrometry (LC/MS) method for long-chain and very-long-chain fatty acid analysis and its application to ¹³C-tracer studies of fatty acid metabolism. Fatty acids containing 14 to 36 carbon atoms are separated by C₈ reversed-phase chromatography using a water–methanol gradient with tributylamine as ion pairing agent, ionized by electrospray and analyzed by a stand-alone orbitrap mass spectrometer. The median limit of detection is 5 ng/mL with a linear dynamic range of 100-fold. Ratios of unlabeled to ¹³C-labeled species are quantitated precisely and accurately (average relative standard deviation 3.2% and deviation from expectation 2.3%). In samples consisting of fatty acids saponified from cultured mammalian cells, 45 species are quantified, with average intraday relative standard deviations for independent biological replicates of 11%. The method enables quantitation of molecular ion peaks for all labeled forms of each fatty acid. Different degrees of ¹³C-labeling from glucose and glutamine correspond to fatty acid uptake from media, de novo synthesis, and elongation. To exemplify the utility of the method, we examined isogenic cell lines with and without activated Ras oncogene expression. Ras increases the abundance and alters the labeling patterns of saturated and monounsaturated very-long-chain fatty acids, with the observed pattern consistent with Ras leading to enhanced activity of ELOVL4 or an enzyme with similar catalytic activity. This LC/MS method and associated isotope tracer techniques should be broadly applicable to investigating fatty acid metabolism.