Aim:Spinal muscular atrophy (SMA) is an inherited, autosomal recessive neuromuscular disease that causes high morbidity and mortality. The prevalence is 1-2/100,000, while the incidence is 1/6000-1/10,000 among live births. Due to the high carrier frequency (1/40-1/60) of SMA, screening can prevent new cases. The aim of the current study was to present the development of a new, quantitative, real-time, polymerase chain reaction (PCR)-based screening test that uses an intelligent ratio (IR) for analyses, as well as a comparison of the results with the gold standard. Materials and Methods:Included in the study were 100 patients with various risk genotypes for survivor motor neuron 1 (SMN1) andSMN2genes whose genetics had been previously investigated using multiplex ligation probe amplification (MLPA). A combination of the 5 ' nuclease assay and allele-specific PCR was used to quantify the SMN1 deletion mutation with real-time PCR using theFIIgene as a reference. All of the optimized standards were adapted to software that provided automated analyses. The approval number of institutional ethics committee for the study is 2012-KAEK-15/1497. Results:The results of the screening test were completely compatible with the MLPA results; it had achieved 100% sensitivity and specificity compared with the gold standard. The use of the IR in the analyses provided a user-independent method that quickly and accurately provided results, regardless of the amount of DNA used of the extraction method. Conclusion:Carrier or newborn screening of SMA is essential in countries that have high rates of consanguineous marriages. The screening test presented in this study that usesFIIas a reference gene proved to be low-cost, reliable, applicable, accurate, and amenable to use in an automated system for SMA screening.