Potency testing of diphtheria and tetanus vaccines traditionally relied on in vivo protection models involving challenge of laboratory animals with toxins while lot release of many other vaccine products is based on serological assays. Here we describe the results of simultaneous serological potency determination of diphtheria (D), tetanus (T) and acellular pertussis (aP) antigens obtained following immunization of guinea pigs with multicomponent pediatric and booster vaccines from different manufacturers. The 4th World Health Organization (WHO) International Standard (IS) for diphtheria toxoid (No. 07/216) and the 4th WHO IS for tetanus toxoid (No. 08/218) were used as reference preparations. For aP, a pediatric vaccine batch containing the antigens pertussis toxoid, filamentous hemagglutinin, pertactin and fimbriae proteins type 2/3 was established as internal control. Quantification of IgG against D, T and aP antigens in guinea pig sera was performed using a hexaplex electrochemi-luminescence immunoassay. We further provide proof-of-concept using experimental vaccine samples lacking or containing reduced amounts of diphtheria toxoid in the presence of full amounts of tetanus and pertussis antigens and alum adjuvant. Importantly, the assay confirmed dose-response relationships for all antigens tested and was able to detect subpotent batches. The results confirmed the suitability of the protocol for combined serology batch release testing of DTaP combination vaccines. This report summarizes the data and the protocol used for validation prior to implementation of this method in routine batch release testing of DTaP vaccines, which led to replacement of in vivo challenge experiments in our laboratory following the 3R (replace, reduce, refine) principle.