Hippotherapy has been used for decades and its benefits to human patients have largely been proven, whether being applied to those with physical or mental disabilities. There have been a plethora of animal welfare studies recently, pertaining especially to ridden horses. This study aimed to investigate stress markers in horses during hippotherapy sessions to address the ethical considerations raised by using horses for therapy. A ridden stress ethogram was established and validated specifically for this study via subjective observation and video recording of a ridden session involving intermediate-level riders. The experiment entailed eight healthy horses undergoing two ridden sessions on separate days, one with disabled riders and one with beginners. Several parameters associated with physiological responses to stress were evaluated at rest, such as heart rate, plasma adrenocorticotropic hormone [ACTH], serum and salivary cortisol. These parameters as well as the behavioural stress score from the ethogram scale were measured during both sessions. No significant differences were found between heart rate, plasma ACTH, and stress scores. Serum and salivary cortisol were significantly lower during the hippotherapy session than during the session with beginners. The current study found no evidence of compromised welfare when horses were used as a therapeutic aid during hippotherapy sessions compared to their usual ridden activity. Although these results indicate that hippotherapy may be ethically justified as it benefits humans without causing harm to the horses, the present study was small, and the results should be interpreted with caution.