To understand and manipulate the interactions between plants and microorganisms, sterile seeds are a necessity. The seed microbiome (inside and surface microorganisms) is unknown for most plant species and seed-borne microorganisms can persist and transfer to the seedling and rhizosphere, thereby obscuring the effects that purposely introduced microorganisms have on plants. This necessitates that these unidentified, seed-borne microorganisms are removed before seeds are used for studies on plant–microbiome interactions. Unfortunately, there is no single, standardized protocol for seed sterilization, hampering progress in experimental plant growth promotion and our study shows that commonly applied sterilization protocols for barley grains using H₂O₂, NaClO, and AgNO₃ yielded insufficient sterilization. We therefore developed a sterilization protocol with AgNO₃ by testing several concentrations of AgNO₃ and added two additional steps: Soaking the grains in water before the sterilization and rinsing with salt water (1% (w/w) NaCl) after the sterilization. The most efficient sterilization protocol was to soak the grains, sterilize with 10% (w/w) AgNO₃, and to rinse with salt water. By following those three steps, 97% of the grains had no culturable, viable microorganism after 21 days based on microscopic inspection. The protocol left small quantities of AgNO₃ residue on the grain, maintained germination percentage similar to unsterilized grains, and plant biomass was unaltered. Hence, our protocol using AgNO₃ can be used successfully for experiments on plant–microbiome interactions.