A method is described for the determination of Ampicillin (α-amino-benzyl penicillin) based on the spectrophotometric measurement at 320 mµ of a compound formed by acid degradation of the penicillin at 75° C in the presence of buffer solution at pH 5·2 and a trace of a copper salt. Results obtained on samples of Ampicillin, Ampicillin Trihydrate and Ampicillin Sodium show good agreement with those recorded by the cup-plate microbiological method.