The extensive presence of large (high molecular weight) protein aggregates in biopharmaceutical formulations is a concern for formulation stability and possibly safety. Tests to screen large aggregate content in such bioformulations are therefore needed for rapid and reliable quality control in industrial settings. Herein, non-commercial seasonal influenza split-virus vaccine samples, produced using various strains and extracted from selected industrial processing steps, were used as model complex bioformulations. Orthogonal characterization through transmission electron microscopy, UV-Vis absorption spectroscopy, fluorescence emission spectroscopy, high-performance liquid chromatography and single-radial immunodiffusion revealed that large, amorphous protein aggregates are formed after virus splitting and their presence is linked mainly, albeit not only, to surfactant (Triton X-100) content in a sample. Importantly, the presence of large virus aggregates in purified whole virus samples and large protein aggregates in vaccine samples was found to correlate with broadening/shouldering in Nile Red fluorescence spectra. Accordingly, decomposition of Nile Red spectra into components allowed the development of a novel, rapid, reliable and user-friendly test with high-throughput potential for screening large aggregate content in influenza split-virus vaccines. The test can be adapted for screening other complex biopharmaceutical formulations, provided relevant controls are done for informed decomposition of fluorescence spectra into their components. (C) 2017 Elsevier Ltd. All rights reserved.