Although the efficiency of retrovirus-mediated gene delivery can be enhanced by several physicochemical approaches reported (e.g., addition of polycations and spinoculation), systematic analysis of retroviral transduction combined with experimental data remains to be challenged. With the aid of a reasonable mathematical description of an experimental system, we can therefore predict and optimize the retroviral gene delivery on a quantitative basis of understanding. In this study, we formulated a mathematical model involved with diffusion, decay and uptake of retroviral vectors onto the target cells resided on a solid culture surface. The model was solved analytically by the Laplace transform method. The analytical solutions were then fitted with experimental data to compute two unknown parameters: concentration of infectious retrovirus and transduction rate constant. Our results showed that the concentration of infectious retrovirus determined by the titration method was approximately hundred-fold lower than the one calculated by fitting experimental data with the mathematical solutions. More importantly, effects of polycation (i.e., Polybrene) on ex vivo retroviral transcluction were illustrated in a quantitative way by estimating the transduction rate constant, which represents a more reliable parameter to determine the degree of transduction of a retroviral vector to a given target cell.