The most critical step in the production of diphtheria vaccines is the inactivation of the toxin by formaldehyde. Diphtheria toxoid (DTx) is produced during this inactivation process through partly unknown, chemical modifications of the toxin. Consequently, diphtheria vaccines are difficult to characterise completely and the quality of the toxoids is routinely determined with potency and safety tests. This article describes the possibility of monitoring the quality in diphtheria vaccine production with a selection of physicochemical and immunochemical tests as an alternative to established in vivo tests. To this end, diphtheria toxin was treated with increasing formaldehyde concentrations resulting in toxoid products varying in potency and residual toxicity. Differences in the quality of the experimental toxoids were also assessed with physicochemical and immunochemical techniques. The results obtained with several of these analyses, including SDS-PAGE, primary amino group determination, fluorescence spectroscopy, circular dichroism (CD) and biosensor analysis, showed a clear correlation with the potency and safety tests. A set of criteria is proposed that a diphtheria toxoid must comply with, i.e. an apparent shift of the B-fragment on SDS-PAGE, a reduction of primary amino groups in a diphtheria molecule, an increased resistance to denaturation, an increased circular dichroism signal in the near-UV region and a reduced binding to selected monoclonal antibodies. In principle, a selected set of in vitro analyses can replace the classical in vivo tests to evaluate the quality of diphtheria toxoid vaccines, provided that the validity of these tests is demonstrated in extensive validation studies and regulatory acceptance is obtained.