AbstractComplement component 4 (C4) is an important plasma protein playing a major role in the human defense mechanism against infectious diseases and inflammatory processes. The C4A and C4B genes, encoding the two isoforms of complement 4, are located in the nuclear serine/threonine protein kinase‐C4A or B gene‐cytochrome 21‐hydroxylase‐tenascin X module (RP‐C4‐CYP21‐TNX) and manifested by variable copy numbers among individuals between zero to six in the human diploid genome. Quantification of the C4A and C4B genes has great clinical importance since unbalanced production of C4A and C4B proteins might be associated with pathological immune processes. Albeit, high‐throughput analysis methods for C4 gene dosage determination are not yet available. Here we present a novel combination of allele‐specific PCR and CGE separation for rapid quantification of the C4A and C4B genes where a single‐step, single‐tube PCR reaction generates two allele‐specific (C4A and C4B) and two control amplicons, followed by CGE analysis of the four fragments. The method presented in this paper enables automated and high‐throughput gene dosage analysis of large sample cohorts.