While a variety of active and passive techniques have been proposed for steady flows, pulsatile flow has received much less attention. Pulsation makes more control parameters available for passive methods and enables them to separate particles. The purpose of this work is to determine the effects of phase shift between two entering flows (only one includes the particles) on particle separation inside a double Y-microchannel. Numerical simulations were carried out for both steady and pulsating flow conditions. The results showed that when the velocity amplitude ratio (β) is less than 2, the separation index increases with the phase shift (φ) and the highest efficiency occurs at φ = 180°. A similar trend can be observed for higher values of β only if the pulsation period is short enough. A series of experiments qualitatively validated the numerical results.