Abacavir (ABC) is an antiretroviral drug highly effective in the treatment of HIV, but its intake can cause severe hypersensitivity reaction (HSR). A strong association between HLA-B*57:01 and ABC HSRs was reported by several studies, which demonstrated that HLA-B*57:01 screening had a 100% negative predictive value and that it could accurately identify patients at high risk of ABC HSRs. We propose a new sequence-specific primer PCR assay based on fluorescence detection through CE which is highly sensitive, allowing the use of non-infective sources of DNA such as saliva and buccal swabs, in addition to blood and reproducible, allowing automation of the analytical process. The results of our study were first compared with a standard sequence-specific primer PCR technique and reported a concordance of 100%, and then a blind external validation further confirmed the accuracy of our method.