The use of probiotics in animal nutrition to provide health benefits is widely accepted. Bifidobacterium animalis (BAN) is an example of a commonly used beneficial strain. BAN is applied in a multi-strain feed additive for poultry. As part of the increased demand for tracking and tracing of feed additives within modern quality management, it is crucial to determine the quantity of the active strain after mixing the probiotic product into feed. A real-time PCR protocol, already developed some years ago, was replaced with a Droplet Digital PCR (ddPCR) assay, as this third generation PCR method is known for higher precision, sensitivity and does not require standard curves. Each sample is partitioned into thousands of small subsamples that are measured individually and an absolute result value for each sample is extrapolated via Poisson distribution. The following parameters were evaluated for the ddPCR assay: optimal annealing temperature (59 °C), concentration of primers (500 nM) and probe (400 nM), and PCR cycle number (50 cycles). The linearity of the optimised ddPCR assay was tested with BAN DNA extracted from pure culture. The obtained standard curve was linear (R2 = 0.9982) and the efficiency (E) of the method was 99.98%. To finalise the development, the Limit of Blank (LoB = 9.17 × 102 copies g-1), Limit of Detection (LoD = 1.15 × 103 copies g-1) and Limit of Quantification (LoQ = 1.57 × 103 copies g-1) for the assay were determined using poultry feed free of BAN and feed spiked with different concentrations of the strain. A BAN strain-specific, probe-based ddPCR assay for the quantification in poultry feed was developed.