Ply steer is an inherent property of a belted tire, manifesting as the nonzero side force at a slip angle of zero, and it is thus an important design factor relevant to a vehicle's straight running, safety, and comfort. Although ply steer is a rolling contact phenomenon, there is a lack of approaches for direct measurement and modeling of tire ply steer force in motion. Thus, we developed an in situ measurement method for tire ply steer based on a recently proposed accelerometer-based intelligent tire system. This new measurement method is significant for three main reasons: It facilitates understanding of the inherent mechanism of ply steer for radial tires, it improves the intelligent tire's accuracy based on the accelerometer, and it provides an in situ measurement approach for tire ply steer. An accelerometer-based intelligent tire was developed to obtain acceleration measurements at different conditions in which the lateral behavior is of particular interest. Two unusual phenomena that have never been reported are observed: (1) lateral accelerations demonstrate asymmetrical behavior with respect to positive/negative slip angles and (2) lateral accelerations at zero slip angle still exist. It is hypothesized that the underlying reason for these two unusual phenomena is the ply steer-dependent kinematics, which can conversely be used to measure tire ply steer in situ. To this end, the mixed Lagrange-Euler approach for rolling contact kinematics is used to formulate tire lateral acceleration, which links both rigid motion and elastic displacement. It is clearly found that the lateral acceleration is proportional to the square of the rotating velocity and the second-order gradient of displacement. Based on this kinematic model, the features of lateral acceleration can be easily explained by tire ply steer, which manifests as the sinusoidal lateral deformation modes due to the coupling of the bending-twist deformation of the cord-rubber composites of the tire belt. The proposed hypothesis has been verified by finite element method simulations, and the experimental results prove that tire ply steer leads to the observed unusual lateral acceleration pattern. Thus, the quantitative value of ply steer could be measured in situ by integrating the obtained lateral acceleration and thus the apparent elastic slip angle, even under zero external wheel slip angle. In this manner, the intelligent tire system provides a direct measurement approach for tire ply in motion. In addition, the accuracy of the intelligent tire's algorithm might be improved by suitable modeling of the asymmetrical acceleration with respect to left/right slip based on the proposed hypothesis of ply steer-dependent kinematics.