Objective The nucleoside diphosphate linked moiety X (Nudix)-Type motif 15 (NUDT15) enzyme is involved in thiopurine metabolism. Genetic variants in the NUDT15 gene result in decreased NUDT15 activity, which in addition to decreased thiopurine S-methyltransferase (TPMT) activity, contributes to thiopurine toxicity. Current standard approaches of NUDT15 genetic analysis have mainly been targeting several common variants. We aimed to develop a clinical-grade DNA-based assay for genetic analysis of the NUDT15 gene using Sanger di-deoxy sequencing. Results Sanger sequencing results were fully concordant with the expected NUDT15 genotype in all 17 cell line samples with known NUDT15 variants (accuracy = 100%; 95% CI 80.49 to 100.00%). Precision studies showed 100% intra-run repeatability and 100% inter-run reproducibility, respectively. Genetic analysis of the NUDT15 gene was performed for 80 patients of Asian ethnicity with wildtype TPMT. 76% (N = 61) of the studied individuals had NUDT15 *1/*1 diplotype. 25% (N = 14) of Chinese and 36% (N = 5) of Malays were found to carry at least 1 non-functional NUDT15 allele. Our study confirmed a high frequency of NUDT15 c.415C>T and c.55_56insGAGTCG variants in the Chinese and Malay ethnic groups in Singapore, highlighting the importance of determining NUDT15 genotype prior to thiopurine dosing.