Abstract: "Pain negatively impacts animal welfare and it is still neglected in ruminants. This original study aimed to develop and validate the Bovine Pain Scale (BPS) for acute pain assessment in hospitalized cattle undergoing surgery. This was a blinded, randomized, prospective clinical study. Thirty-six animals were included in the study. The Pain Group (n = 25) included patients admitted to a veterinary teaching hospital requiring any type of soft tissue or orthopedic surgery. Videos were recorded before, 2–6 hours after surgery, 1 hour after the administration of analgesia and 24 hours after surgery. The Control Group (n = 11) included healthy animals that were video recorded twice within a 24-48h interval. The BPS was developed using content validity. A total of 118 videos of 6 minutes were randomized and analyzed by four raters who were unaware of groups, time-points and procedures in two phases with a five-week interval. Statistical analysis was performed using R software. Intra and inter-rater reliability (intra-class correlation coefficient) was very good (0.83–0.94) and ranged from good to very good, respectively (0.65–0.81). The correlation between the BPS and the Visual Analog Scale (VAS) was strong (rho = 0.77, p < 0.0001) confirming criterion validity. Item-total correlation was acceptable for 3 of 9 items (0.33–0.43) and internal consistency was below the acceptable value (0.6). The scale was responsive to pain but not the administration of analgesia. It was specific for five items, but no items showed sensitivity. The area under the curve of 0.90 demonstrated high discriminatory capacity. According to the receiver operating characteristic curve, the cut-off point for rescue analgesia was ≥ 5 of 18. The BPS is reliable and reproducible, showed content and criterion validity, and may be used in veterinary hospitals for assessing post-operative pain in cattle to guide decision-making towards rescue analgesia. Future studies should refine the instrument to guarantee construct validity and sensitivity."