Establishing effective cage-side pain assessment methods is essential if post-surgical pain is to be controlled effectively in laboratory animals. Changes to overall activity levels are the most common methods of assessment, but may not be the most appropriate for establishing the analgesic properties of drugs, especially in mice, due their high activity levels. Use of drugs that can affect activity (e.g. opioids) is also a problem. The relative merits of both manual and automated behaviour data collection methods was determined in two inbred mouse strains undergoing vasectomy following treatment with one of 2 buprenorphine dose rates. Body weights and the effects of surgery and buprenorphine on faecal corticosterone were also measured. Surgery caused abnormal behaviour and reduced activity levels, but high dose buprenorphine caused such large-scale increases in activity in controls that we could not establish analgesic effects in surgery groups. Only pain-specific behaviour scoring using the manual approach was effective in showing 0.05 mg/kg buprenorphine alleviated post-vasectomy pain. The C57 mice also responded better to buprenorphine than C3H mice, indicating they were either less painful, or more responsive to its analgesic effects. C3H mice were more susceptible to the confounding effects of buprenorphine irrespective of whether data were collected manually or via the automated approach. Faecal corticosterone levels, although variable, were higher in untreated surgery mice than in control groups, also indicating the presence of pain or distress. Pain-specific scoring was superior to activity monitoring for assessing the analgesic properties of buprenorphine in vasectomised mice. Buprenorphine (0.01 mg/kg), in these strains of male mice, for this procedure, provided inadequate analgesia and although 0.05 mg/kg was more effective, not completely so. The findings support the recommendation that analgesic dose rates should be adjusted in relation to the potential severity of the surgical procedure, the mouse strain, and the individual animals' response.