Flagellins are the main structural proteins of bacterial flagella and potent stimulators of innate and adaptive immunity in mammals. The flagellins of Salmonella are virulence factors and protective antigens, and form the basis of promising vaccines. Despite broad interest in flagellins as antigens and adjuvants in vaccine formulations, there have been few advances towards the development of scalable and economical purification methods for these proteins. We report here a simple and robust strategy to purify flagellin monomers from the supernatants of liquid growth culture. Phase 1 flagellins from Salmonella enterica serovars Typhimurium (i epitope) and Enteritidis (g,m epitopes) were purified directly from conditioned fermentation growth media using sequential cation- and anion-exchange chromatography coupled with a final tangential flow-filtration step. Conventional porous chromatography resin was markedly less efficient than membrane chromatography for flagellin purification. Recovery after each process step was robust, with endotoxin, nucleic acid and residual host-cell protein effectively removed. The final yield was 200-300 mg/L fermentation culture supernatant, with similar to 45-50% overall recovery. A final pH 2 treatment step was instituted to ensure uniformity of flagellin in the monomeric form. Flagellins purified by this method were recognized by monoclonal anti-flagellin antibodies and maintained capacity to activate Toll-like Receptor 5. The process described is simple, readily scalable, uses standard bioprocess methods, and requires only a few steps to obtain highly purified material. (C) 2014 The Authors. Published by Elsevier Inc.