Empirical observations generally indicate a shifting and decreased Lolium spp. susceptibility to glyphosate in Italy. This is likely due to the long history of glyphosate use and to the sub-lethal doses commonly used. There is, therefore, a need to determine the variability of response of Lolium spp. to glyphosate and identify the optimum field dose. To perform a sensitivity analysis on Lolium spp. populations in an agriculture area, collection sites were mainly chosen where glyphosate had not been applied intensely. Known glyphosate-resistant or in-shifting populations were included. Two outdoor dose-response pot experiments, including eleven doses of glyphosate, were conducted. The dose to control at least 93%–95% of susceptible Lolium spp. was around 450 g a.e. ha−1. However, to preserve its efficacy in the long term, it would be desirable not to have survivors, and this was reached at a glyphosate dose of 560 ± 88 g a.e. ha−1. Taking into account the variability of response among populations, it was established that the optimal dose of glyphosate to control Lolium spp. in Italy up to the stage BBCH 21 has to be at least 700 g a.e. ha−1. As a consequence, it is recommended to increase the label recommended field rate for Lolium spp. control in Italy to a minimum of 720 g a.e. ha−1.