Abstract Interferon (IFN)-γ ELISPOT can be used to monitor the magnitude of virus-specific cellular immune responses in vaccine trials. Often, IFN-γ ELISPOT is performed with cryopreserved peripheral blood mononuclear cells (PBMC). However, it has not been well defined yet to what extent diminished cell viability of PBMC following cryopreservation affects IFN-γ responses in ELISPOT. Therefore, we assessed the influence of apoptotic cells on the number of spot-forming cells (SFC) in IFN-γ ELISPOT using a gradient of UV-irradiated apoptotic PBMC and viral antigens derived from varicella zoster virus (VZV) and cytomegalovirus (CMV). No SFC were observed when UV-irradiated apoptotic cells were stimulated with VZV or CMV antigens. Moreover, presence of apoptotic cells among viable T cells hampered the detection of SFC following stimulation with VZV or CMV cell lysates, but not with CMVpp65 peptide pool. Statistical analysis showed that mainly late apoptotic cells, staining both Annexin V and 7-amino-actinomycin D (7-AAD), were associated with a decreased number of SFC. In conclusion, it is recommended to use highly viable thawed PBMC for the detection of virus-specific cellular immune responses by IFN-γ ELISPOT, since the detection of CMV- and VZV-specific T cell responses stimulated by cell lysates was significantly impeded by the presence of apoptotic cells.