Small hydrocyclones are an attractive technology for biomass separation from fermentation processes. The interactive effect of design parameters on the performance of mini-hydrocyclones is, however, not fully explored and studies are often limited by the challenges in manufacturing such small units. Here, 10-mm mini-hydrocyclones are produced by 3D printing and the impact of spigot diameter, vortex finder diameter and height on separation performance is studied. A central composite rotatable design was adopted to obtain information on the relation between the variables and their influence on concentration ratio and recovery of yeast from a highly diluted system. A Pareto front for separation performance was generated and shown to be suitable to select an optimal design for a set of process constraints.