Flocculation unit operations are being revisited as a strategy to ease the burden posed on clarification and purification operations by the increasingly high cell density cultures used in the biopharmaceutical industry. The purpose of this study was to determine the key process parameters impacting flocculation scale-up and use this understanding to develop an automated ultra-scale down (USD) method for the rapid characterization of flocculation at the microliter scale. The conditions under which flocculation performance of a non-geometrically similar vessel three orders of magnitude larger can be mimicked by the USD platform are reported. Saccharomyces cerevisiae clarified homogenate was flocculated with poly(ethyleneimine) (PEI) to remove the residual solids remaining in the centrate. Flocculant addition time modulated flocculation performance depending on the predominant mixing time scale (i.e. macro-, meso- or micromixing). Particle growth and breakage was mimicked at the two flocculation scales by the average turbulent energy dissipation (ϵavg) and impeller tip speed (vtip) scale-up bases. The results obtained were used to develop an USD method. The USD method proposed uses constant ϵavg as the scale-up basis under a micromixing controlled regime. These conditions mimicked the STR flocculation performance within a ±5% error margin. Operation in the mesomixing regime led to particle size deviations between the flocculation scales of ≤50 %. These results, in addition to the microscopic observations made, demonstrate the USD system presented in this work can produce process-relevant flocculated material at the microliter scale under the correct operating conditions. Biotechnol. Bioeng. 2014;111: 2486–2498. © 2014 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.