Hereditary α tryptasemia (HαT) is an autosomal dominant trait characterized by increased TPSAB1 copy number (CN) encoding α-tryptase. The determination of HαT is being discussed as an important biomarker to be included in risk assessment models and future diagnostic algorithms for patients with mastocytosis and anaphylaxis. Due to the complex genetic structure at the human tryptase locus, genetic testing for tryptase gene composition is presently notably limited and infrequently pursued. This study aimed to develop, optimise and validate a multiplex droplet digital PCR (ddPCR) assay that can reliably quantify α- and β-tryptase encoding sequences in a single reaction. To optimise the ddPCR conditions and establish an amplitude-based multiplex ddPCR assay, additional primers and probes, a thermal gradient with varying annealing temperatures, different primers/probe concentrations, and various initial DNA quantities were tested. Results obtained from all 114 samples analysed using multiplex ddPCR were identical to those obtained through the use of original duplex assays. Utilizing this multiplex ddPCR assay, in contrast to conducting distinct duplex ddPCRs, presents noteworthy benefits for tryptase genotyping. These advantages encompass a substantial threefold decrease in material costs and considerable time savings. Consequently, this approach exhibits high suitability and particularly captures interest for routine clinical implementation.