Saponins from Quillaja saponaria have been commonly used as immunomodulatory adjuvants in foot‐and‐mouth disease vaccines (FMDVs). However, due to the lack of consensus over the possible exacerbation of local inflammatory responses in cattle and its economic impacts, their use has been discouraged by Brazilian authorities. A qualitative method intended to determine the presence of saponins from Q. saponaria bark extracts in FMDVs was developed and validated. Instrumental analysis was performed using an liquid chromatography (LC) coupled to a quadrupole–time‐of‐flight–mass spectrometry (TOF‐MS) system. The method was validated according to the International Conference on Harmonization Harmonized Tripartite Guideline Q2 (R1) and Brazilian Ministry of Agriculture, Livestock and Food Supply Analytical Quality Assurance Guidelines. Validation parameters were determined and considered suitable to the established criteria. The validated method has been applied in routine analysis in the National Agricultural Laboratory at Rio Grande do Sul (LANAGRO‐RS). All results obtained were in agreement with the vaccine's composition described by the manufacturer. The method is easy and adequate for analysis in routine laboratories. To the best of the authors' knowledge, this is the first report of a method which intends to investigate the presence of saponins from Q. saponaria bark extracts in veterinary vaccines.