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Water is the main factor for the healthy life of plant. One of the main negative effects of climate change is the increasing scarcity of water that is lethal for plant. Globally, for water deficit regions (arid and semi-arid), drought is the main factor responsible for low production of agriculture, especially for cotton. Great efforts have been and are being made to find alternatives to water saving practices. This study aimed to examine the effects of partial rhizosphere drying (PRD, half of the root system irrigated at one event, and the other half irrigated in the next event, and so on) with and/or without various mulching treatments on physiological and biochemical traits of cotton. To explore this objective, we laid out experiments in completely randomized design with factorial arrangement in the Islamia University of Bahawalpur, Pakistan in 2016. Two factors included were four mulching treatments (M0, no mulching; M1, black plastic mulching; M2, wheat straw mulching; and M3, cotton sticks mulching) and two irrigation levels (I0, control (full irrigation); and I1, PRD). Fisher’s analysis of variance among means of treatments was compared using least significant difference test at 5% probability level. Results revealed that the maximum plant height, leaf area, leaf gas exchange (photosynthetic rate and stomata conductance), chlorophyll, proline and total sugar contents, and enzyme activities were higher under M2 than under other three mulching treatments. As for irrigation levels, higher values of plant height, photosynthesis and water related parameters (leaf water potential, leaf osmotic potential, leaf turgor potential, etc.) were recorded. Contents of total sugar and proline and activities of antioxidant enzymes were significantly higher in PRD-treated plants than in control plants. It was concluded that combined application of PRD and mulching was more effective than the rest of the treatments used in the experiment. Similar study can be conducted in the field by applying irrigation water in alternate rows in semi-arid regions.