Weeds incur significant losses to the crops plants but not only competing with crops for water, nutrients, sunlight, and space but also harbor insects and diseases. The herbicide resistant cotton offers farming community an opportunity to combat weeds by providing flexibility of herbicide application whenever needed. In present study, the herbicide resistant cotton was evaluated to unravel the glyphosate dose response at different time intervals. Glyphosate was applied on advance transgenic cotton line VH-289 at the time intervals of 20, 25, 30 and 40 days after germination and compared with manual weeding of its conventional non-transgenic counterpart as control. The weed control efficacy, types, mortality, and population of nineteen different weeds was calculated with reference to the time and dose of glyphosate spray (600 mL.acre ^-1, 800 mL.acre ^-1, 1000 mL.acre ^-1, 1200 mL.acre ^-1, 1400 mL.acre ^-1, 1600 mL.acre ^-1, 1800 mL.acre ^-1 and 2000 mL.acre ^-1) and its impact on yield. The yield and contributing characters like boll weight and cotton yield were higher in lines where glyphosate was applied at 20 and 30 days after germination compared to other treatment (manually hoed). Importantly, weeds control efficiency was recorded up to 98% at a dose concentration of 1800 mL.acre ^-1 and 2000 mL.acre ^-1 after 15-30 days after glyphosate application (DAS). The average number of bolls per plant in glyphosate resistant line was recorded higher compared to non-transgenic manually hoed control. This study highlights that spraying 1800 and 2000 mL.acre ^-1 glyphosate can help to eradicate weeds in field at less cost and good yield return. The results can be helpful for the cotton farmers regarding the proper concentration of glyphosate and its time of application.