© 2022 Author(s).Passive cell separation methods have attracted great attention due to their superiority over the other methods stemming from their easy fabrication, precise manipulation, cost-effectiveness, sensitivity, and simplicity. The fluid inertia in these methods is the main factor that is affected by the channel design; thus, the channel design parameters should be chosen accordingly. Even though all channel design parameters are well addressed in inertial microfluidics, the curvature angle of the channel has not yet been extensively studied. In this study, three different curvilinear microchannels with curvature angles of 180°, 210°, and 270° were designed, keeping all other remaining parameters the same. The focusing ability of the fluorescent polystyrene microparticles with diameters of 1.1, 3.3, and 9.9 μm was investigated both experimentally and numerically to understand focusing efficiency affected by the curvature angle of the microchannel. The first set of experiments was to determine the effect of the channel curvature and indicated the favorable design as channel C, which showed focusing qualities of 0.85 and 0.92 for 9.9 μm particles at volumetric concentrations of 2% and 5%, respectively. The remaining set of experiments and CFD simulations were conducted to observe the interaction of 3.3 and 9.9 μm particles and reveal the distortion of the focusing line and particulate phase contours for 9.9 μm particles at the flow rates between 0.3 and 0.7 ml/min, which was further confirmed by enriched mixtures containing 1.1, 3.3, and 9.9 μm particles. The study showed that mixtures comprising low diameter particles could not satisfy the focusing criteria, which emphasized the importance of an appropriate particle size and concentration for a single focus line. On the other hand, it was shown that geometric features of the microchannel such as the hydraulic diameter and the curvature angle together with the particle size determine the focusing quality both experimentally and numerically. To sum up, the increment of the channel curvature angle is a determining factor for particle focusing, and a single focusing line was observed on the particles maintaining the focusing criteria even in many particle conditions. While the focusing quality of the particles was reduced by multi-particle interactions, they were proven to be separable achieving the appropriate concentration ratio.