Aromatic L-amino-acid decarboxylase (AADC) deficiency is an inherited disorder of biogenic amine metabolism with a broad phenotypic spectrum including motor dysfunction and autonomous dysregulation during the first year of life. Since the clinical phenotype shows some overlap with other diseases the diagnosis and start of therapy are usually delayed. Innovative disease-changing treatment options, particularly gene therapy, have highlighted the need for early diagnosis. Here we demonstrate that 3-O-methyldopa (3-OMD) in dried blood spot is a reliable marker for high-through-put newborn screening. We established a novel tandem mass spectrometry method to quantify 3-OMD in dried blood spots (DBS) of 38,888 unaffected newborns, and successfully tested it in 12 heterozygous carriers, six known AADC-deficient patients, and 1,079 healthy controls at different age groups. Reference ranges for 3-OMD concentrations in 38,888 healthy newborns revealed a mean of 1.16 µmol/l (ranging from 0.31 to 4.6 µmol/l). Non-AADC control subjects (n= 1,079) from 0 to 18 years showed a mean 3-OMD concentration of 0.78 µmol/l (ranging from 0.24 to 2.36 µmol/l). There was low intra- and inter-assay variability and 3-OMD concentrations were stable over a period of 32 days under different storage conditions at -20 °C, 4 °C, and 21 °C. With a cut-off value set at 5 µmol/l we identified six confirmed AADC-deficient patients with a mean of 13.15 µmol/l 3-OMD (ranging from 3 to 36.93 µmol/l), yet to a lesser extent and a negative correlation with age. Therefore, further age-dependent reference values of 3 µmol/l (age 28 days to 10 years) and 2 µmol/l (age > 10 years) were defined. 3-OMD concentrations of 12 carriers revealed a mean of 0.72 µmol/l (ranging from 0.46 to 1.09 µmol/l). 3-OMD concentrations appeared neither age- nor sex-dependent. We demonstrate a novel high-throughput method to measure 3-OMD in newborn screening DBS, which allows integration in existing newborn screening programs to enable early diagnosis of AADC deficiency.