Objectives The purpose of this pilot study was to evaluate the use of an intramuscular (IM) sedation protocol with butorphanol and alfaxalone in cats undergoing blood donation. We hypothesized that this drug combination would provide sufficient sedation to perform phlebotomy without causing hypotension or significant changes in heart rate. Methods Six purpose-bred, healthy adult cats were sedated using IM butorphanol (0.4 mg/kg) and alfaxalone (2-3 mg/kg). Pulse and Doppler blood pressure (BP) were recorded at baseline, after sedation and immediately following phlebotomy. Once laterally recumbent, 12 ml/kg blood was collected from the jugular vein. Sedation scores, duration of lateral recumbency and the ability to successfully perform phlebotomy were recorded. Results There was no significant change in heart rate post-sedation (median 190 beats per min [bpm], range 160-224 bpm) or post-phlebotomy (median 200 bpm, range 180-220 bpm) compared with baseline values (median 200 bpm, range 180-220 bpm) ( P = 0.395). A statistically significant change in BP was detected ( P = 0.029), attributed to a difference between post-sedation (median 113.3 mmHg, range 110.7-130.0) and baseline (median 133.3 mmHg, range 130.0-183.3) measurements. Hypotension was not observed in any cat. Collection of at least 80% of the target volume was achieved in 5/6 cats, although all were adequately sedated to allow jugular venous phlebotomy. Median recumbency time was 53 mins (range 43-83 mins). Phlebotomy duration lasted a median of 13 mins (range 5-21 mins). Conclusions and relevance The administration of IM alfaxalone and butorphanol provided sufficient restraint for blood donation without causing hypotension or significant changes in heart rate before or after phlebotomy.