A prospective, randomized, crossover experimental pilot study was performed to compare the anesthetic quality and recovery times of intramuscular (IM) ketamine/medetomidine and S-ketamine/medetomidine in Testudo spp. Four adult male Hermann's tortoises (Testudo hermanni) and one adult male spur-thighed tortoise (Testudo graeca) were used in this study. Each animal underwent two different anesthetic protocols twice, with a washout period of two weeks in between each anesthetic event. This resulted in 20 anesthetic procedures. Protocols consisted of 0.1 mg/kg medetomidine IM with either 15 mg/kg ketamine (KM) or 15 mg/kg S-ketamine (SKM). After 60 min, medetomidine was reversed using 0.5mg/kg atipamezole IM. Muscle relaxation, ease of handling, sensitivity to a painful stimulus, palpebral and corneal reflexes, heart rate (HR), and respiratory rate (RR) were measured every 15 min during anesthesia. Time of initial effect, maximal anesthetic effect, first movements, complete wakefulness, first feeding after anesthesia, and quality of induction and recovery periods were compared between protocols. Quality of induction and recovery were excellent with both protocols. HR decreased within the first 15 min but began to rise at 60 min. Marked bradypnea occurred at the beginning of anesthesia but gradually disappeared over time. Muscle relaxation and handling scores increased during anesthesia, whereas sensitivity scores remained 0, regardless of protocol. Pairwise comparisons showed no significant differences between KM and SKM, with the exception of the palpebral reflex at 30 min (P < 0.046). Anesthetic quality was similar using KM or SKM. Both protocols were safe and effective in Testudo spp., but analgesic effects were considered weak.