Mitochondrial fatty acid beta-oxidation (FAO) disorders are clinically and biochemically heterogeneous diseases mainly associated with intolerance to catabolic stress. These disorders can now be detected pre-symptomatically by newborn screening, and thus the clinical phenotype in an individual patient may be unclear. Correlation of clinical severity with concentrations of acylcarnitine species was investigated in fibroblasts from FAO-deficient patients presenting with various phenotypes and asymptomatic neonates detected by newborn screening. Intact cells were incubated in medium containing deuterium-labelled hexadecanoic acid and L-carnitine for 72h, and the accumulated acylcarnitines in the culture medium analysed using electrospray tandem mass spectrometery. Fibroblasts from patients with long-chain FAO disorders presenting at an early age and with poor clinical outcomes accumulated higher concentrations of long-chain acylcarnitine species compared with those from patients with milder phenotypes. This suggests that the in vitro quantitative acylcarnitine profiling could perhaps predict the prognosis of some FAO defects. This would be particularly useful information for the asymptomatic/pre-symptomatic FAO-deficient infant detected by the expanded newborn screening program, in whom the risk of developing symptoms later in life is not known.