Glucose-6-phosphate dehydrogenase (G6PD) deficiency holds critical health concerns, particularly due to its association with drug-induced hemolysis triggered by medications such as antimalarials. This condition poses significant risks in malaria-endemic regions where the prevalence and genetic diversity of G6PD deficiency further complicate management. Providing accurate and reliable G6PD status is vital to ensuring safe treatment, reducing complications, and improving healthcare outcomes in these populations. This study evaluated the integration of phenotypic and genotypic diagnostic methods for identifying G6PD deficiency in 2953 participants in Thailand. Using the water-soluble tetrazolium salts enzymatic assay and multiplex high-resolution melting analysis, the study revealed an overall prevalence of 3.93%, with 7.19% in males and 1.83% in females. A total of 38 distinct G6PD genotypes were identified, and zygosity was determined, highlighting significant genetic diversity, including previously unreported mutations as identified by sequencing. Hemizygous males, homozygous females, and approximately 50% of heterozygous females with missense mutations exhibited deficient or intermediate phenotypes. However, 40% of females carrying G6PD missense mutations showed a normal phenotype in quantitative phenotypic testing. The findings highlight the need for accurate G6PD diagnosis to improve drug safety and efficacy, particularly for vulnerable individuals such as heterozygous females, who are at risk of hemolysis. The cost-effective, high-throughput methods demonstrated here are suitable for large-scale screening, making them especially valuable in resource-limited settings. To maximize their impact, integrating both phenotypic and genotypic approaches into national healthcare policies and malaria programs is essential. By ensuring equitable access to reliable G6PD testing, these findings support malaria elimination efforts and address broader healthcare challenges, ultimately reducing preventable morbidity and mortality associated with G6PD deficiency.