Castration is one of the most frequently performed surgical procedures in livestock. All methods of castration are associated with a significant degree of pain, affecting the patients’ welfare. Limited information on species-specific protocols in buffaloes is available. Considering the importance of this species and the scarcity of reports available, the objective of this study was to evaluate the effects of two different intramuscular doses of xylazine for premedication in water buffalo calves undergoing isoflurane maintenance anaesthesia for routine castration surgery. Twenty clinically healthy male water buffaloes undergoing bilateral orchiectomy were randomly assigned to two groups of 10 animals each: Group 1 was premedicated with xylazine 0.1 mg/kg bw i.m. and Group 2 was premedicated with xylazine 0.15 mg/kg bw i.m. Anaesthesia was induced by ketamine (2 mg/kg bw i.v.) and maintained with isoflurane-oxygen-air in both groups. The HR, RR, presence/absence of ataxia, sedation, depth of anaesthesia, muscle relaxation status, response to acoustic and tactile stimuli, eyelid reflex, extent of salivation and stimulus were evaluated every 5 min. Both protocols provided consistent sedation and were safe. Faster and more satisfactory sedation was recorded when xylazine was administered at 0.15 mg/kg bw i.m., leading us to recommend this dose for buffalo orchiectomy.