In this study alfalfa (Medicago sativa L.) were grown in a standard medium fortified with different inorganic selenium species in a pot experiment, and then harvested upon vegetation 4 times during the year of 2019. The aim of this experiment was to understand the physiology of alfalfa when they accumulated selenium in different concentrations and to determine the selenium species present in the plant using a chromatographic separation coupled with an inductively coupled plasma mass spectrometry detection. Moreover, to prove the reproducibility of the seleno-amino acids in alfalfa from the first enriched harvest to the end of its growing cycle, which is important for producing Se-enriched forage. The benefit of Se-enriched alfalfa adds nutritional value to the leaf protein concentrates and can be used as a cheap dietary source for animal feed.