Abstract: Measuring thyroxine levels from dried blood samples using mass spectrometry. A test sample is provided that was obtained by treating a dried blood sample with an extraction solution. The test sample also includes an isotopically enriched thyroxine standard. The test sample is scanned using a mass spectrometer to produce one or more mass spectra and the level of thyroxine in the test sample is determined by comparing a peak in the one or more mass spectra that corresponds to thyroxine with a peak in the one or more mass spectra that corresponds to isotopically enriched thyroxine. The level of thyroxine in the dried blood sample is optionally determined based on the extraction efficiency of the extraction solution. Thyroxine levels may be measured in combination with amino acid and/or carnitine levels. Compositions and kits for practicing the method are also provided.