The objectives of this study were to establish a donkey ethogram, followed by a donkey grimace scale to be applied to donkeys pre- and post-castration and to test if there was a notable difference in scores based on observer knowledge, gender, and experience, which could reveal possible discomfort/pain. Nine healthy male adult donkeys were surgically castrated. Fifty-four photos were selected from frontal, lateral, and body views taken pre- and post-castration. Observers ranging from minimal to extensive knowledge and levels of experience based on education and hours/month spent with donkeys scored six photos/donkey on a scale of 0–2 (0 = not present, 1 = moderately present, 2 = obviously present). Scores were based on body language and facial parameters: Ears down, ears back, eye white showing, glazed look, orbital tightening, eyes round shape, nostril tension, eyes narrow shape, muzzle tension, and abnormal stance and overall perception of the animal being in pain. Level of experience and knowledge, as well as gender significantly (p < 0.001), affected observers’ ability to accurately score images. The study suggests that the most significant indicators of pain in donkeys are overall appearance and abnormal body stance provided their sensitivity, specificity and accuracy values of 63.18%, 62.07%, and 62.60%, respectively.