Abstract Aims To provide a reliable, reproducible and centrifuge-free filtration protocol for clarification of large volumes of bacterial cultures. Methods and Results Four experiments were designed to compare different techniques enabling clarification of Escherichia coli cultures using as a benchmark the concentration and quality of bacterial outer membrane vesicles (OMVs). The experiments were designed to examine the performance of different extraction methods on large volume (≥1 L) filtrations of bacterial culture media. Performance parameters included filtration flow rates, sterility testing and characterization of the filtrates by: (i) SDS-PAGE, (ii) cryogenic transmission electron microscopy, (iii) nanoparticle tracking analysis and (iv) Qubit protein quantification. The experiments revealed that: (i) addition of the filter aid Diatomaceous Earth to the bacterial cultures improved filtration flow rates significantly and eliminated the need for centrifugation prior to filtration; (ii) sterile filtration was successful as no bacterial passage was identified through the membrane filter; (iii) centrifuge-free filtrates contained an increased amount of OMVs compared to centrifuged filtrates. Conclusions In comparison to conventional centrifuge-based protocols, the clarification method presented has universal applicability for a broad range of microbial extraction procedures, regardless of the volume of culture harvested. Moreover, the decreased amount of OMVs presented in the filtrates following centrifugation step provides an additional argument in favour of a centrifuge-free approach. Significance and Impact of the Study Sterile filtration is a universal method for the clarification of bacterial cultures. Common challenges related to filtration include filter clogging and long processing times, due to limited centrifugation capacity, which can affect product quality. The proposed protocol is likely to ensure a highly effective filtration process and could be a novel approach in improving the filtrate products without the need of centrifugation.