Aromatic L-amino-acid decarboxylase (AADC) deficiency is an inherited disorder of biogenic amine metabolism resulting in severe neurologic impairment. The diagnosis is often delayed due to unspecific symptoms and the need for complex diagnostic tools. With new treatment options, particularly gene therapy, pre-symptomatic diagnosis is more im- portant than ever. We established and tested a novel high-throughput method to quantify 3-O-methyldopa (3-OMD) in dried blood spots using electro- spray-ionization mass spectrometry (ESI-MS/MS). We measured 38,888 newborns, 14 heterozygous DDC variant carriers, seven AADC deficient patients, as well as 1079 healthy control subjects to validate the method. 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 deficient 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 deficient patient. Intra- and inter-assay variability was low. We demonstrate an innovative ESI-MS/MS method to quan- tify 3-OMD in DBS with extremely short measurement time and low cost, enabling early diagnosis of AADC deficiency in a high-throughput new- born screening approach for the first time.