Abstract: An enzymatic process for the determination of .beta.-lactam antibiotics in a biological liquid, comprising the steps of (1) incubating the liquid with soluble D-alanyl-D-alanine-carboxypeptidase produce by Actinomadura R39, the .beta.-lactam antibiotic reacting with the enzyme to form an inactive and equimolecular enzyme-antibiotic complex; (2) incubating the mixture obtained with a solution of a thioester substrate having the formula ##STR1## wherein R.sub.1 =benzoyl, phenylacetyl or N.sup..alpha. -acetyl-L-lysyl,R.sub.2 =glycyl or D-alanyl, andR.sub.3 =hydrogen or methylso as to form, by hydrolysis, a 2-mercaptoalkanoic acid having the formula ##STR2## in an amount proportional to the residual enzymatic activity, said incubation being further conducted in the presence of glycine or of a D-amino acid; (3) determining the amount of 2-mercaptoalkanoic acid formed, and (4) comparing the determination of step (3) with a standard, to obtain the antibiotic concentration in the biological liquid.