Introduction: Allopurinol is a most widely used uric-lowering agent, especially in patients with renal impairment or underwent chemotherapy. Although mild maculopapular eruption occurred in 2% of patients, severe cutaneous adverse drug reactions (SCAR), such as Steven-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and drug reaction with eosinophilia and systemic symptoms (DRESS) are very rare (approximately 0.4%). However, their mortality rates are high, up to 30%, and re-introduction of the medication is contraindicated. The T lymphocyte and various cytokines, such as tumour necrosis factor (TNF)- and interferon (IFN)- are accepted as the key mediator in most delayed drug reactions. Many in vitro tests have been used in diagnosis of mild delayed drug reaction, but very limited studies in SCAR. Objective: To validate IFN- ELISpot in diagnosis of allopurinol-induced SCAR, and to compare IFN- producing cells (spots) between PBMC of patients who experienced allopurinol-induced SCARs and tolerant patients. Methods: Heparinized blood were collected from a patient who has experienced SCARs. PBMC were prepared, using Ficoll-Hypaque technique. Culture media were complete RPMI-1640 containing 10% Fetal Bovine Serum (FBS) and penicillin-streptomycin 100 u – 100 μg/ml RPMI. PBMC of 3 x 105 cells per well were cultured with or without drug (allopurinol or oxypurinol 0.5, 1, 5, 10, 20 or 50 μg/ml) for 18 hours 6 (overnight). Allopurinol was dissolved using 0.001 M NaOH. PHA was used as positive control. IFN- producing cells were detected using automated KS ELISpot reader. Aged-match healthy control and allopurinol-tolerant patients were used as control group. All experiments were performed in triplicate. Results: In all 3 allopurinol-induced SCAR (2 SJS and 1 DRESS), IFN- producing cells, using NaOH as drug solvent in allopurinol-SJS were higher than allopurinol-tolerant and heatlhy subjects at all drug concentration and unfortunately at baseline (no drug stimulation) No significant increase in spots was detected between baseline and after drug stimulation in allopurinol-SCAR patients. Conclusions: IFN- ELIspot is a sensitive tool in detection of T cell response. Although, our experiments were not be able to detect a significant difference in number of IFN- producing cells before and after drug stimulation in allopurinol-SCAR patients. This method should be repeated in a larger sample size. Combination of multiple functional tests, such as cytotoxicity assay, granulysin detection, and lymphocyte proliferation test using flow cytometry, might be useful.