An enzyme-linked immunosorbent assay for the detection of mustard protein was developed. The assay is based on a polyclonal antiserum directed against a mixture of mustard proteins raised in rabbits. The assay has a detection limit of 1.5 ppm (milligrams per kilogram) and is suitable for the detection of traces of mustard protein in mustard seed-derived flavoring ingredients. Limited cross-reactivity testing showed that no other plant proteins reacted significantly. From the animal proteins tested, only milk showed some cross-reactivity. With this sensitive assay, it was shown that refined mustard seed oil produced by steam distillation does not contain detectable amounts of mustard protein. Mustard seed oil is used as a flavoring in very low quantities, typically between 40 and 200 mg/kg. Thus, 100 g of a food product flavored with 200 mg of mustard seed oil per kg containing <1.5 mg of protein per kg would represent an amount of mustard seed protein of <30 ng. Taking into account the published literature on allergic reactions to the unintended ingestion of mustard, this conservatively low calculated level indicates that it is unlikely that food products containing mustard seed oil as a flavoring ingredient will elicit an allergic reaction in mustard-allergic individuals. Copyright ©, International Association for Food Protection.