Existing therapies for glioblastoma multiforme do not ensure patient’s recovery. Oncolytic viruses (OV) represent a promising alternative as they can destroy glioblastoma-initiating stem cells, which the major cause of relapses. However, while individual OV strains are effective for some patients, they could be ineffective for others. To achieve a predictable therapeutic effect, live tumor cells of the patient need to be tested for the sensitivity to different viruses. The aim of this study was to assess how the sensitivity of tumor cells to viruses changes with passaging in cell culture. Primary glioblastoma cell cultures were prepared from excised tumors. We compared sensitivity of cells to four non-pathogenic enteroviruses (type 1 poliovirus, Coxsackie virus A7, Echoviruses 1 and 12), for freshly-explanted primary tumor cell cultures and for those that had undergone 700 divisions during the passaging. The sensitivity was assessed based on the proportion of viable cells by the MTT assay 72 hours after the cells were inoculated with serial 10-fold dilutions of virus preparations. Cells isolated from the tumors of 3 patients exhibited varying sensitivity to the used viral strains. Differences in the lowest virus dose required for the successful infection of cell cultures were as high as 105. Passaging induced sensitivity shifts, such as increased or decreased sensitivity to individual viruses. Differences in the sensitivity correlated with the ability of the infected cells to produce the virus. Based on our findings, we conclude that the sensitivity of cancer cells to viruses should be tested at very early stages of passaging, preferably in primary cultures.