Several pharmacogenetic studies have revealed strong associations between specific human leukocyte antigen (HLA) alleles and the susceptibility to drug hypersensitivity. Recently, we reported HLA-B*3505 as a strong genetic biomarker for the nevirapine-induced skin rash in Thai population. Here, we developed a new HLA-B*3505 genotyping method by a combination of the Universal Invader assay and sequence-specific primer PCR. From the sequence alignment of 68 HLA-B alleles in the Thai population, we selected the two most discriminative SNPs (rs1140412 and rs4997052) as target SNP sites. When we carried out the assay using 324 Thai individuals, fluorescence intensities of HLA-B*3505-positive and HLA-B*3505-negative samples were apparently discriminated at the endpoint of the reaction. Our results were 100% concordant with those obtained by a sequence-based typing method. As our assay is simple and rapid, we believe our method will be a useful tool for pharmacogenetic testing of the nevirapine-induced skin rash.