Background Drug-induced severe cutaneous adverse reactions (SCARs) are presumed T-cell-mediated hypersensitivities associated with significant morbidity and mortality. Traditional in-vivo testing methods, such as patch or intradermal testing, are limited by a lack of standardisation and poor sensitivity. Modern approaches to testing include measurement of IFN-γ release from patient peripheral blood mononuclear cells (PBMC) stimulated with the suspected causative drug. Objective We sought to improve ex-vivo diagnostics for drug-induced SCAR by comparing enzyme-linked immunospot (ELISpot) sensitivities and flow cytometry-based intracellular cytokine staining (ICS) and cellular composition of separate samples (PBMC or blister fluid cells (BFC)) from the same donor. Methods IFN-γ release ELISpot and flow cytometry analyses were performed on donor-matched PBMC and BFC samples from four SCAR patients with distinct drug-allergies. Results Immune responses to suspected drugs were detected in both PBMC and BFC samples of two donors (Case 1 in response to ceftriaxone and Case 4 to oxypurinol), with BFC eliciting stronger responses. For two other donors, only BFC samples showed a response to meloxicam(Case 2) or sulfamethoxazole and its 4-Nitro metabolite (Case 3). Consistently, flow cytometry revealed a greater proportion of IFN-γ-secreting cells in the BFC compared to PBMC. BFC cells from Case 3 were also enriched for memory/activation/tissue-recruitment markers over PBMC. Conclusion Analysis of BFC samples for drug-allergy diagnostics offers a higher sensitivity for detecting positive responses compared to PBMC. This is consistent with recruitment (and enrichment) of cytokine-secreting cells with a memory/activated phenotype into blisters.