Introduction: Aromatic L-amino-acid decarboxylase (AADC) defciency is an inherited disorder of biogenic amine metabolism resulting in severe neurologic impairment. The diagnosis is ofen delayed due to unspecifc symptoms and the need for complex diagnostic tools. With new treatment options, particularly gene therapy, pre-symptomatic diagnosis is more important than ever. Methods: We established and tested a novel high-throughput method to quantify 3-O-methyldopa (3-OMD) in dried blood spots using electrospray-ionization mass spectrometry (ESI-MS/MS). We measured 38,888 newborns, 14 heterozygous DDC variant carriers, seven AADC defcient patients, as well as 1079 healthy control subjects to validate the method. Results: In 38,888 healthy newborns we measured a mean 3-OMD concentration of 1.16 µmol/l (SD=0.31, range 0.31–4.6 µmol/l), whereas AADC defcient patients showed a mean of 9.88 µmol/l (SD=13.42, range 1.82–36.93 µmol/l) with a negative correlation of 3-OMD concentrations with age. Highest 3-OMD concentrations were detected in a stored NBS card of an AADC defcient patient. Intra- and inter-assay variability was low. Conclusion: We demonstrate an innovative ESI-MS/MS method to quantify 3-OMD in DBS with extremely short measurement time and low cost, enabling early diagnosis of AADC defciency in a high-throughput newborn screening approach for the frst time.