The antiretroviral agent abacavir can cause hypersensitivity reaction (HSR) and the presence of HLA-B*57:01 is predictive of abacavir-HSR in Caucasian HIV-infected patients. However, abacavir-HSR also occurs in HLA-B*57:01 negative patients. In these patients, a safe diagnostic tool to dissect clinically suspected HSR against abacavir from adverse reactions against co-administered drugs is mandatory, and abacavir-ELISpot was evaluated. Peripheral blood mononuclear cells from 87 HIV patients were stimulated by abacavir and the production of interferon-γ to the ELISpot was determined. Abacavir treated patients with HSR [confirmed (n=5) or suspected (n=12)] vs without HSR (n=42) displayed significantly higher numbers of abacavir-specific cells (82.3±23.0 or 10.5±4.5 vs -0.5±1.0 spot forming cells per million PBMC, p < .005 each). In conclusion, we established the first abacavir-specific ELISpot. According to our preliminary data, a negative abacavir-ELISpot nearly excludes HSR against abacavir. Thereby the ELISpot may facilitate the decision to continue or withdraw abacavir treatment.