We established a new 'transmission kinetic assay (TKA)' to quantify the human cytomegalovirus (HCMV) transmission between cells in vitro and to phenotypically characterize HCMV strains based on their mode of transmission by flow cytometric analysis. On one hand we used the genetically modified HCMV strain TB40/E-delUL16-GFP, and on the other hand, clinical isolates. When twofold diluted infecting cells were seeded to a constant number of uninfected cells, the transmission of virus on each day (day 0-5) followed a strictly linear pattern, which was characterized by a linear equation. The slope of this linear equation represents 'the number of newly infected cells per infecting cell'. To standardize the TKA, the slopes of the different days were plotted against the corresponding days. This resulted in a new linear equation with a new slope value, which characterizes the transmission kinetics. To differentiate cell-associated and cell-free modes of transmission, we introduced HCMV neutralizing antibodies into the system. The slope was 0.9 (±0.5) when the virus exhibited only cell-associated transmission and was 4.1 (±0.7) when the virus exhibited both modes of transmission. TKA was then applied to different clinical isolates and they were phenotypically characterized based on their modes of transmission. Apart from the quantitative analysis of HCMV transmission and the phenotypical characterization of clinical isolates, the TKA was applied to quantify the inhibition of clinical isolates transmission by immune cells and to study the effect of cytokine (IL-2) on immune cells inhibiting HCMV transmission.