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Summary: Gastrointestinal helminth infection occurs within a diverse microbiome, complicating the interpretation of whether effects are caused by the parasite versus the microbial community. Here, we present a protocol for deriving sterile larvae of the murine helminth, Heligmosomoides polygyrus bakeri (H. polygyrus), providing experimental control of the microbiome. We describe steps for sterilizing with a bleach solution and developing into infectious larvae using E. coli. We then detail procedures for removing bacterial contaminants before harvesting to ensure the generation of germ-free larvae. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.