Spinal muscular atrophy (SMA) is an autosomal recessive disease caused, in about 95% of SMA cases, by homozygous deletion of the survival motor neuron 1 (SMN1) gene or its conversion to the highly homologous SMN2 gene. The molecular diagnosis of SMA is usually carried out by a PCR-Restriction fragment length polymorphism (RFLP) approach. However, this approach is not useful for identification of healthy deletion carriers. TaqMan technology is one of the most reliable and widely adopted techniques for the SMN1 copy number evaluation. However, several limitations of this technique have been described. Particularly, DNA extraction methods and accurate template quantification have been shown to be critical for reliable results. In this work, we set up a reliable, highly reproducible, and easy-to-perform TaqMan technology-based protocol to obtain the SMN1 gene copy number assessment. We demonstrate that PCR amplification of both target gene and reference gene in the same reaction mix, instead of separated mixes, greatly reduces reported criticisms of simplex TaqMan technology. The multiplex real-time PCR we describe allows interlaboratory samples and data exchange, without the need to equalize the DNA isolation technique. Further, the protocol described below requires fewer replica tests than the simplex methodology does, leading to reduced overall cost for the diagnostic assay.