In this work, we study the influence of inertia on the dynamics of neutrally buoyant spherical microbeads of varying diameter in a Pinch Flow Fractionation device. To that aim, we monitor their trajectory over an unprecedented wide range of flow rates and flow rate ratios. Our experimental results are supplemented by a depth-averaged 2D-model where the flow is described using the Navier-Stokes equation coupled with the shallow channel approximation and where particles trajectories are computed from Newton's second law of motion with a particle tracing model. Above a certain flow rate, we show that particles inertia enables them to cross streamlines in response to an abrupt change of direction. These streamline crossing events combined with the increasing effect of the inertial lift forces drive particles to deviate from the inertialess trajectory. The amplitude of the resulting inertial deviation increases both with the particles diameter and the total flow rate before reaching a plateau. Consequently, based on our numerical and experimental results, we determine the optimal flow conditions to shift the particles distribution to significantly enhance their size-based separation.