Turner syndrome (TS) is one of the most common human chromosomal abnormalities; it is characterized by the presence of one normal X chromosome and the complete or partial loss of the second X chromosome. The early recognition of TS patients allows for adequate therapy for short stature and pubertal sex steroid substitution. We developed a cost-effective molecular diagnostic tool that can be used to identify 45,X TS patients from dried blood spots, for possible use in neonatal screening for TS. We used a three-step method for 45,X TS detection: i) DNA extraction from dried blood spot samples, ii) pre-PCR HpaII digestion (methylation-sensitive enzyme) and iii) GeneScan analysis of selected cases. DAX-1 gene amplification was used to recognize DNA integrity, and the androgen receptor gene (Xq11-12), which is both a highly polymorphic and methylated gene, was used to determine the number of X chromosome alleles. Using this three-step diagnostic procedure, we detected apparent TS in 1/304 (0.33%) samples; such individuals should be submitted to clinical examination and karyotype confirmation. The three-step 45,X TS neonatal screening protocol is a simple, reliable, fast (under 30 h) and cost-effective diagnostic tool, useful for the neonatal detection of TS.