Perennial ryegrass (Lolium perenne) is the predominant forage crop in the equatorial highland zones of Colombia due to its high nutritional value and versatility to produce both milk and meat. This study aimed to determine the relationship between the relative depletion of usable soil water and the Ks values of canopy expansion and closure stomatal of perennial ryegrass, as well as to identify the threshold values of water stress. The experiment was carried out in pots under a controlled environment condition. These pots were arranged in a completely randomized manner. The experiment consisted of five treatments—including control treatment—of water deficits in the soil that progressively increased the depletion level as the crop cycle developed. This generated a wide range of conditions in the growth stages. For each treatment, four repetitions were performed Biomass production was significantly affected by water stress. The results show that the upper and lower thresholds of Ks were 0.28 and 1.3 of the depletion level (p) of the total available water (TAW) in the soil for the expansion of the canopy (CE), and 0.25 and 1.1 p of the TAW for stomatal closure (gs). Quadratic functions were fitted for both the CE (R2 = 0.72) and CS (R2 = 0.73), moreover, the Ks function of FAO-AquaCrop with positive shape factor (sf) was as follows: sf = 11, RMSE 0.22 for CE, and sf = 4.3, RMSE 0.19 for gs. Our results indicate that ryegrass is moderately sensitive to water stress. The differences found between the Ks function of FAO and the experimental data call for the need to use modeling with parameters adapted for each case.