The mitochondrial fatty acid beta-oxidation is integral to normal cellular metabolism and maintenance of cellular energy supplies. Disorders of this pathway interrupt the body's ability to deal with fasting states, as well as compromising the functioning of organs and systems whose high-energy requirements utilize fats for a continuous energy source, such as heart and skeletal muscle. This method quantitatively measures intermediate metabolites of fatty acid beta-oxidation, specifically the 3-hydroxy-fatty acids produced by the third step in the pathway. The method is useful for helping to diagnose disorders of the pathway, especially defects in the L-3-hydroxyacyl CoA dehydrogenases. Serum or plasma samples are used for routine clinical evaluation; however, measurement of 3-hydroxy-fatty acid intermediates in fibroblast cell culture media and in samples from mice also allows the method to be used for research into fatty acid oxidation and interconnected pathways. The method is a stable isotope dilution, electron impact ionization gas chromatography/mass spectrometry (GC/MS) procedure.