The activated slip system of Ti-6Al-4V alloy during the superplastic forming (SPF) was investigated by the in-grain misorientation axes analysis (IGMA), and the mechanisms of slip system activation have been discussed. Depending on the distribution of IGMA, one significant discovery from this study is that all the basal, prismatic, and pyramidal slip systems would be activated. Considering the effective slip systems, Schmid factors, and the Euler angles together, it is suggested that the dominant slip systems not only desired the largest Schmid factors but strongly demand continuous Schmid factors among the adjacent grains. Meanwhile, the estimated critical resolved shear stress (CRSS) on basal <a> and prismatic <a> at the temperature of 920 °C with the strain rate of 10−3 s is given. An original method of roughly estimating dominant slip systems with Euler angles has been introduced, which predicts that grain rotation may change the slip system. Furthermore, the crystal plasticity finite element method (CPFEM) is employed to simulate the evolution of Euler angles, and the grain orientation presents the largest set of significant clusters around the after deformation. Besides, the continuity of the Schmid factor assumption for the activated slip system has also been verified by CPFEM. In addition, the eigenvector corresponding to the eigenvalue λ1 = 1 of Euler angle rotation matrix is calculated to be aligned with the grain rotation axis, which can be applied to describe the grain rotation.