Lentiviral vectors have become popular tools for stable genetic modification of mammalian cells. In some applications of lentiviral vector-transduced cells, infectious-lentiviral particles should be absent. Quantification of the free-vector particles that remain from the inoculum can be difficult. Therefore a formula was established that yields an estimation of the 'Reduction Ratio.' This ratio represents the loss of titer based on a number of vector-inactivating effects. In this study, we evaluated several parameters and assumptions that were used in the current formula. We generated new data on the stability and trypsin sensitivity of lentiviral vectors pseudotyped with eight heterologous envelope proteins and the loss of vectors by washing or passaging the cell cultures. Our data demonstrate that the loss of virus titer under the influence of trypsin as well as the half-life of the particles in tissue culture medium is dependent on the vector's envelope protein. While VSV-G-envelope-pseudotyped particles were unsensitive to trypsin, the titer of vectors pseudotyped with other envelope proteins decreased 2-110-fold. The half-life in culture medium ranged from 8 to 40 h for the different envelope-pseudotyped vectors, with 35 h for VSV-G-envelope-pseudotyped vector particles. Additionally, we found that removal of the culture medium from Ø35 mm to Ø10 cm dishes reduces the amount of vector particles in the culture by 50-fold and 20-fold, respectively. Together these data can be used to more precisely estimate the maximum number of free lentiviral vector particles in cell cultures.