<p>This study investigates inertial particle separation in highly viscous fluids using a microfluidic platform designed for size-selective particle manipulation through combined numerical and experimental approaches. The working fluid viscosity was systematically controlled using water–glycerol mixtures to examine its influence on particle migration behavior. Microchannels incorporating pillar arrays with circular, square, diamond, and triangular geometries were employed to evaluate the effects of local hydrodynamic boundaries on particle–wall interactions. The numerical results were validated through qualitative and quantitative flow visualization using the circular pillar configuration. The results show that pillar geometry has a strong influence on local velocity gradients. Square pillars generate the steepest velocity gradients and the largest high-velocity regions within the inter-pillar gaps, whereas triangular pillars induce asymmetric flow fields and nonuniform velocity distributions. The results also show that fluid viscosity systematically shifts the critical diameter (<i>D</i>_<i>c</i>) governing inertial focusing. Particles smaller than <i>D</i>_<i>c</i> exhibit zigzag trajectories with negligible net lateral displacement, while particles larger than <i>D</i>_<i>c</i> transition to a bumped migration mode. This mode transition enables effective size-based particle separation. These results provide experimentally validated numerical insight into how fluid viscosity and pillar geometry influence inertial microfluidic separation under high-viscosity conditions.</p> Graphical Abstract <p></p>