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.