Natural killer (NK) cells are major components of the innate immune system and are assumed to play an important role in the defense against cytomegalovirus infection. Human cytomegalovirus (HCMV) is the only virus known that shapes the NK cell receptor repertoire in humans. Assays allowing the simultaneous measurement of multiple cell parameters and the assessment of subpopulations of NK cells are suitable for monitoring the NK cell response to infected cells. Herein, we describe an autologous assay system to assess NK cell responses to HCMV-infected macrophages which is based on flow cytometry to detect CD107a expression and interferon gamma (IFNγ) secretion. Further we established a simple method to handle HCMV-infected macrophages. Our assay provides a practicable approach to investigate NK cell responses to HCMV infection in vitro.