In Greece, the National Newborn Screening Program was initiated in 1974 and is performed by the Institute of Child Health (ICH). However, there is a complete absence of conditions that have high rates of mortality and a relatively high prevalence listed in the Catalogue of Disorders screened by the ICH. Our laboratory has expanded the existing NBS program to include newborn screening for inborn errors of metabolism, screening for cystic fibrosis (the most common congenital disorder in the Greek population), congenital adrenal hyperplasia, and for biotinidase deficiency. From July 2007 to December 2009, 45,000 dried blood spots (DBS) were collected from infants born in Athens, Greece, and were analyzed. We present a report of our 30-month experience in the newborn screening area. The samples were tested for amino acidopathies, fatty acid oxidation disorders (FAOD), and organic acid metabolic disorders by applying flow injection analysis-electrospray ionization-tandem mass spectrometry (FIA-ESI-MS/MS); for cystic fibrosis by immunoreactive trypsinogen (IRT) measurement (time-resolved fluoroimmunoassay); for congenital adrenal hyperplasia by fluoroimmunoassay to measure the 17 hydroxy-progesterone level; and for biotinidase deficiency using a colorimetric method and a semiquantitative fluoroimmunoassay to determine biotinidase activity. Sample analysis resulted in establishing cutoff values for the respective disease markers for the first time in the Greek population. Four infants were identified with cystic fibrosis, two with congenital adrenal hyperplasia, two with phenylketonuria (PKU), one with medium-chain acyl CoA dehydrogenase deficiency (MCADD), and one with biotinidase deficiency. To the best of our knowledge, this is the first article reporting the status of expanded newborn screening in Greece.