Introduction: Ergot alkaloids (EAs) are toxic substances naturally produced by Claviceps fungi. These fungi infest a widerange of cereals and grasses. When domestic animals are exposed to EAs through contaminated feeds, it is detrimental to them andleads to significant economic losses. For that reason, it is important to monitor feed for the presence of EAs, especially with methodsenabling their determination in processed materials. Material and Methods: Ergot alkaloids were extracted with acetonitrile, anddispersive solid phase extraction (d-SPE) was used for clean-up of the extracts. After evaporation, the extracts were reconstitutedin ammonium carbonate and acetonitrile and subjected to instrumental analysis using high-performance liquid chromatographywith fluorescence detection. The developed method was validated in terms of linearity, selectivity, repeatability, reproducibility,robustness, matrix effect, limits of quantification and detection and uncertainty. The EA content of 40 compound feeds wasdetermined. Results: All the assessed validation parameters fulfilled the requirements of Regulation (EU) 2021/808. At least oneof the monitored alkaloids was determined in 40% of the samples. The EAs with the highest incidence rate were ergocryptine,ergometrinine and ergocornine. The total concentrations of EAs ranged from under the limit of quantification to 62.3 μg kg−1.Conclusion: The results demonstrated that the developed method was suitable for simultaneously determining twelve EAs incompound feed and could be used for routine analysis