HLA-A*31:01 and HLA-B*15:02 have been widely reported to confer genetic susceptibility to carbamazepine (CBZ)-induced severe cutaneous adverse reactions (SCARs). Accordingly, the screening for these alleles has been highly recommended to prevent SCAR prior to introducing CBZ therapy. Although a number of methods are available for screening of HLA-A*31:01 or HLA-B*15:02 alleles separately, developing an assay that can detect both these alleles would be more clinically practical, cost-effective and less time-consuming. Therefore, in this study, a multiplex polymerase chain reaction (PCR) using TaqMan Probe was designed and validated to be able to detect HLA-A*31:01 and HLA-B*15:02. In comparison with Luminex-SSO/SBT/SSB, the multiplex PCR assay for detection of HLA-A*31:01 and HLA-B*15:02 had a perfect agreement in the validation group of 125 samples. The method was able to detect the target genes at the DNA concentration of 0.037 ng/μL. The unit cost of this assay is less than $5 USD with total time of 110 minutes.