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Study to find out the response of wheat (Triticum aestivum L.) cultivars to exogenous application of 100 mM glycinebetaine (GB) at different growth stages (vegetative, flowering and grain filling) was carried out under water limited environment, at the Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad during 2008-09, to find out the best GB application stage for improvement in drought tolerance potential. The wire house experiment was laid out in completely randomized design. Data regarding various physiological and biochemical parameters of crop were recorded using standard procedures. The data so collected were analyzed statistically by using the Fisher’s analysis of variance technique and LSD at 5% probability was used to compare the differences among treatment means. Drought stress at all three critical growth stages adversely affected plant’s nutrient uptake and it also reduced the net photosynthesis rate (Pn) and transpiration rate (E) of wheat plant. Exogenous application of GB under drought at all three critical growth stages improved tolerance of wheat by reducing toxic nutrient’s uptake however, grain filling stage was found more responsive.