Substances with cytotoxic activity present in vaccines against the foot-and-mouth disease may interfere with methods used to detect residual live virus in the product or cause undesirable postvaccination reactions. This study describes a rapid in vitro test to detect cytotoxic activity in water-in-oil vaccines against foot-and-mouth disease using a commercial saponin as a cytotoxic agent and a solution of sheep’s red blood cells as substrate. Hemolytic and cytotoxic activity was analyzed using experimental and commercial vaccines prepared with and without saponin. The hemolytic and cytotoxic potential of preparations containing saponin was evident. In contrast, hemolytic and cytotoxic activities were not observed in vaccines without saponin in their composition. The method described here allows to easily detect if the vaccine under study has cytotoxic activity, making it possible to select the most appropriate method to process the sample to be used for the innocuity test. Additionally, due to undesirable effects that may be observed in animals receiving vaccines containing saponin in their formulation, the use of the rapid test described here allows to identify those vaccines with cytotoxic activity and to verify the presence of saponin on them, through the mass spectrometry method.