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Background: Vaccination is widely used in fish aquaculture for three primary reasons: to prevent bacterial disease spreading, minimize antibiotic use and fight antibiotic resistance. Vaccine production is an expensive and consuming process, mainly in terms of money, resources and animals for quality control. The 3Rs (Replace-Reduce-Refine) philosophy suggests developing and validate alternative methods to animal testing for scientific purpose, even for biologicals and vaccines. Aim: The current study explored the potential use of mouse and fish cells in the in vitro toxicity grade assessment thorough different methods, as alternative assay to in vivo Residual Toxicity Test for autogenous fish vaccines control. Methods: BF2 and L929 cell lines were exposed to vaccines dilutions in two different ways of administration and toxicity grade was recorded by MTS assay, in comparison to the in vivo gold standard test. Results: AVs caused no reactions in the in vivo test. In the in vitro assay, the different toxicity grade recorded was statistically significant between the cell lines adopted and the AVs way of administration. Conclusion: Data obtained represent the first application of 3Rs method to fish autogenous vaccines produced in Italy, more investigations are needed to collect solid results and standardize new in vitro methods for vaccines quality control. [Open Vet J 2023; 13(4.000): 495-500]