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Canine rabies is responsible for an estimated 59,000 human deaths every year. In an attempt to reach the ZeroBy30 goal, robust disease surveillance coupled with improved diagnostics play a paramount role in ensuring reliable data and gradually attesting rabies control advancements. In this context, proficiency testing is organized to harmonize rabies diagnostic capacities. In most exercises, rabies-positive samples consist of brains collected from intracerebrally inoculated mice. This procedure causes distress and severe suffering to animals, raising important ethical concerns that can no longer be ignored. In the last decades, the 3Rs tenet (Replace, Reduce, Refine) has been successfully implemented in several scientific areas, and we strongly support its application in the framework of rabies proficiency testing. Here, we discuss cell-based technologies as innovative sustainable in vitro candidate systems to replace in vivo experiments for the production of proficiency testing samples. The application of these alternative methods can allow completely in vitro or ex vivo production of rabies proficiency testing panels, which would represent an important replacement or reduction/refinement for current in vivo procedures.