Obtaining sterile anticoagulated whole-blood units is a key prerequisite for downstream laboratory and clinical applications involving blood-derived products. In bovine medicine, however, published information on blood collection into bags is mainly embedded in transfusion-oriented references or broader downstream studies, while formal validation of donor-oriented collection methods remains limited. The aim of this study was to develop and field-validate a large-volume bag-based method for whole-blood collection in donor dairy cows under commercial farm conditions. Whole-blood collections were performed in clinically healthy donor dairy cows using a jugular venipuncture protocol that was progressively refined during field validation. A true sequential multi-bag setup, enabled by extension tubing and a perforable connection device, was introduced on 24 October 2022. Protocol validation was based primarily on procedural success, defined as a bacteriologically negative blood unit with a collected volume of at least 400 mL per bag. A total of 294 procedures were included. Overall, 228/294 procedures (77.6%) were classified as successful, 48/294 (16.3%) as suboptimal, and 18/294 (6.1%) as failed. The median collected volume per procedure was 410 mL (IQR 400–430; range 124.5–1,280 mL). Overall, 276/294 collections (93.9%) were bacteriologically negative. Within the single-bag phase, later collection dates were associated with a lower probability of bacteriological positivity, supporting a learning-curve effect on microbiological reliability. Later implementation of the sequential multi-bag setup was associated with a higher total volume per procedure, without an apparent loss of microbiological safety. In conclusion, this large-volume bag-based protocol proved feasible for whole-blood collection in donor dairy cows under field conditions and may provide a practical upstream basis for subsequent laboratory applications and downstream processing workflows.