Being able to obtain an objective and repeatable measurement of stress levels from a single subject represents a primary issue in animal welfare science, especially in relation to cattle farming. A potential solution has been recently identified in the determination of cortisol’s salivary levels: a non-invasive method strictly related to acute stress. The development of an on-field, easy-to-use method to perform the analysis is yet to be validated. In this study, we developed and tested the reliability of salivary cortisol as a marker for the evaluation of animal welfare. We aimed to develop an on-field use Lateral Flow Immunoassay (LFIA) for the evaluation of acute stress in bovines. The availability of this non-invasive diagnostic tool will facilitate monitoring of animal welfare. In the short term, this innovation is expected to assist farmers and veterinarians in performing a more objective evaluation of the animal’s acute stress levels. In the long term, the device could become a key-instrument for the EU’s growing necessity to better monitor and identify the potential stressor factors in animal farming.