Herein, we present the synthesis, origin and characterization of potential impurities of Ampicillin trihydrate, viz. Ampicillinyl-d-phenylglycine (Ph. Eur impurity E) & l-Ampicillin (Ph. Eur impurity B). The impurity Ampicillinyl-d-phenylglycine originates during the preparation of Ampicillin trihydrate, amidation of carboxylic acid of Ampicillin with d-phenylglycine generates Ampicillinyl-d-phenylglycine, the impurity l-Ampicillin may arise due to the presence of traces l-phenylglycine methylester in d-phenylglycine methylester hydrochloride which may result in the formation of l-Ampicillin. A facile synthetic process for these impurities was developed from the commercially available raw materials.