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Introduction: The Cytomegalovirus (CMV) is the major cause of morbidity and mortality in solid organ (SOT) and bone marrow (BMT) transplantation. An early reconstitution of immune response is crucial in limiting CMV replication; on the other hand, a late reconstitution may determine CMV reactivation with possible evolution to symptomatic phase.The Enzyme-Linked Immunosorbent Spot (EliSPOT) assay is a useful tool for monitoring the CMV-specific immune response recovery in transplant patients.This study propose the development and optimization of a interferon-gamma-(IFN-γ)-based EliSPOT assay for the detection of CMV immune response. Methods: CD3 + lymphocytes were separated using Robosep (negative selection, HLA ® EasySep WB Human T Cell Enrichment Kit, Stemcell Technology,Vancouver, Canada).The EliSPOT assay was optimized using the Human IFN gamma ELISPOT Kit and a modified protocol provided by Nanogen Advanced Diagnostics (Buttigliera Alta, Italy). Different parameters were analyzed: number of cells (200,000 - 300,000), antigens (CMV peptide mix and whole CMV antigen), incubation time (18 - 20 - 22 - 24h), number of washes, incubation conditions with secondary antibody and conditions of substrate development. Results: Herein we report the optimal conditions identified. 200,000 CD3+ cells per well were stimulated with CMV peptide mix antigens. Cells were stimulated for 18h at 37 °C in humidified atmosphere; wells were washed and secondary antibody was added and incubated at room temperature for 2h.After incubation a second series of washes were performed. Substrate was added and incubation for 15 minutes was carried out.The reaction was detected by plate reader AID ELISPOT (Strassberg, Germany). Conclusions:We developed an EliSPOT assay for the detection of CMV-immune response and reconstitution.All variables were compared to previous published protocols.The improvement of the EliSPOT assay for the detection of CMV-specific immune response represents the first step for result evaluation in clinical practice.