BACKGROUNDAlfalfa (Medicago sativa L) is one of the most planted crops worldwide primarily used to feed animals. The use of alfalfa in human diet as sprouts, infusions and nutritional supplements is rapidly gaining popularity. Despite this, allergenicity assessment of this novel plant food is largely lacking.RESULTSHere, leaf protein extract of alfalfa was studied using a combined proteomics, Immunoglobulin E (IgE)‐binding inhibition assay and in silico approach to find potential allergens. We have identified and annotated 129 proteins using in‐gel digestion proteomics and Blast2Go suit. A search against COMPARE database, using the identified proteins as query sequences, revealed high similarity with several allergenic proteins. The Single Point Highest Inhibition Achievable assay (SPHIAa) performed on the multiplex FABER® allergy testing system confirmed the in silico results and showed some additional potential allergens. This approach allowed the detection of proteins in alfalfa leaves cross‐reacting with plant allergens from three different allergen families such as lipid transfer, thaumatin‐like and Bet v 1‐like protein families. In addition, the absence of structural determinants cross‐reacting with seed storage allergenic proteins and with animal allergens was recorded.CONCLUSIONThis study reports for the first time potential allergenic proteins in alfalfa. The results suggest that this plant food can be safely introduced, as a protein‐rich supplement, in the diet of patients allergic to animal food allergens. Allergic patients towards certain plant food allergens need to be careful about consuming alfalfa because they might have allergic symptoms. © 2020 Society of Chemical Industry