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Patients with glucose-6-phosphate dehydrogenase deficiency might develop acute hemolytic anemia, chronic hemolytic anemia, and neonatal hyperbilirubinemia when exposed to high levels of oxidative stress. Severe hemolysis may occur in not only patients but also female carriers under certain conditions. However, 80%–85% of female carriers were undetected in an existing newborn screening program because of their wide-ranging levels of enzyme activity. We developed a cost- and time-efficient multiplex SNaPshot assay using dried blood spots. By detecting 21 common mutations in Taiwan and Southeast Asia, the assay could determine 98.2% of the mutant alleles in our cohort of Taiwanese newborns. The 9 undetermined mutant alleles were consequently detected by Sanger sequencing, of which 5 unpublished variations—c.187G > A (Pingtung), c.585G > C (Tainan), c.586A > T (Changhua), c.743G > A (Chiayi), and c.1330G > A (Tainan-2)—were detected. Furthermore, 13% of mild mutations were missed in male infants whose enzyme levels at 6.1–7.0 U/gHb in the newborn screening program when set the cutoff value at 6.0 U/gHb. We therefore suggest increasing the cutoff value and applying the multiplex SNaPshot assay as the second tier for neonatal screening. Our approach could significantly increase the detection rate of male patients and female carriers with a reasonable cost and a reasonable number of clinic referrals. • We propose a cost- and time-efficient multiplex SNaPshot assay using dried blood spots. • With the 21 G6PD mutations selected, the assay could determine 98.2% of mutant alleles in Taiwan. • We strongly suggest applying the proposed assay to the current newborn screening program. • The proposed assay could increase detection rates and control clinic referrals. [ABSTRACT FROM AUTHOR]