The feasibility of using nanocellulose-based mille-feuille filter paper for upstream applications in serum-free growth media filtration, i. e. Dulbecco's modified Eagle's medium (DMEM) and Luria-Bertani medium (LBM), was tested. The filter performance with respect to F.174 bacteriophage (28 nm) removal as a model smallsize virus was characterized for filters of varying thicknesses, i. e. 11 and 33 mu m, at varying operating pressures, i. e. 1 and 3 bar. The filters demonstrated generally good model small-size virus removal properties with LRV = 5, especially for 33 mu m filters. The 33 mu m filters were more robust and exhibited better virus removal and throughput properties than 11 mu m filters, although their flux was generally lower. The performance of the 33 and 11 mu m nanocellulose-based filter papers was further verified for upscaled bioprocessing by 10-fold increase in the loading volume. The results of the present work show that the 33 mu m nanocellulose-based filter paper could be an interesting alternative for large volume cell culture medium filtration during upstream bioprocessing.