Inertial focusing plays a major role in size-based cell separation or enrichment for microfluidic applications in many medical areas such as diagnostics and treatment. These applications often deal with suspensions of different particles which cause interactions between particles with different diameters such as particle-particle collision. In this study, particle-particle interaction in a laminar flow through a low aspect ratio alternating and repetitive microchannel is investigated both numerically and experimentally. It is revealed that particle-particle collision affects high quality particle focusing. computational fluid dynamics simulations are conducted for demonstrating the effect of the flow field in the transverse cross-section on the focusing quality and position. The experiments and simulations both revealed that if the flow is seeded with a mixture of particles of 3.3 and 9.9 mu m diameters, the quality of focusing intensity is degenerated compared to the focusing features obtained by particles with a diameter of 9.9 mu m solely. The results clearly show that particle-particle collision between the 3.3 and 9.9 mu m particles has a negative effect on particle focusing behavior of the 9.9 mu m particles.