This study investigates the effect of room temperature deformation on the microstructural behavior of Ti6Al4V alloy. To study this, room temperature uniaxial compression test was carried out at low strain rate of 0.01 s-1 with increase in 5% stepwise degree of deformation up to fracture. At each stage of the deformation, Stress strain curve was correlated with change in microstructure .Microstructural evolution and grain Fragmentation are mapped at each stage of deformation with the help of EBSD and OPTICAL microscopy. Fracture of material occurs within 30% of deformation and exhibit grain refinement. Flow stress increases with increase in deformation and indexed in terms of Increase in strain hardening exponent and hardness. EBSD mapping and Microstructural analysis confirms Alpha phase fragmentation and grain size reduction.