Abstract Background B chromosomes are classified as dispensable genomic components tolerated by cells, which are transmitted to progeny despite providing no benefit in most cases. They have been observed in over 2800 species of plants, animals and fungi, including numerous maize accessions. As maize is one of the most important crops worldwide, research on the maize B chromosome has been pioneering in the field. The characteristic of the B chromosome is its irregular inheritance. This results in offspring with a different number of B chromosomes compared to the parents. However, the exact number of B chromosomes in the studied plants is a crucial piece of information. Currently, assessing the number of B chromosomes in maize largely depends on cytogenetic analyses, which are laborious and time-consuming. We present an alternative approach based on the droplet digital PCR technique (ddPCR), which is faster, more efficient and provides the results within one day with the same level of accuracy. Results In this study, we report a rapid and straightforward protocol for determining the number of B chromosomes in maize plants. We developed a droplet digital PCR assay using specific primers and a TaqMan probe for the B-chromosome-linked gene and a single-copy reference gene on maize chromosome 1. The performance of the assay was successfully verified by comparison with the results of cytogenetic analyses performed in parallel. Conclusions The protocol significantly improves the efficiency of B chromosome number assessment in maize compared to cytogenetic approaches. The assay has been developed to target conserved genomic regions and can therefore be applied to a wide range of diverged maize accessions. This universal approach can be modified for chromosome number detection in other species, not only for the B chromosome but also for any other chromosome in aneuploid constitution.